Investigation into the Molecular Mechanisms underlying the Anti-proliferative and Anti-tumorigenesis activities of Diosmetin against HCT-116 Human Colorectal Cancer
Investigation into the Molecular Mechanisms underlying the Anti-proliferative and Anti- tumorigenesis activities of Diosmetin against HCT-116 Human Colorectal Cancer
Mohammed A. Alshawsh
Published: xx xx xxxx Diosmetin (Dis) is a bioflavonoid with cytotoxicity properties against variety of cancer cells including hepatocarcinoma, breast and colorectal (CRC) cancer. The exact mechanism by which Dis acts against CRC however, still remains unclear, hence in this study, we investigated the possible molecular mechanisms of Dis in CRC cell line, HCT-116. Here, we monitored the viability of HCT-116 cells in the presence of Dis and investigated the underlying mechanism of Dis against HCT-116 cells at the gene and protein levels using NanoString and proteome profiler array technologies. Findings demonstrated that Dis exhibits greater cytotoxic effects towards HCT-116 CRC cells (IC50 = 3.58 ? 0.58 ?g/ml) as compared to the normal colon CCD-841 cells (IC50 = 51.95 ? 0.11 ?g/ml). Arrests of the cells in G2/M phase confirms the occurrence of mitotic disruption via Dis. Activation of apoptosis factors such as Fas and Bax at the gene and protein levels along with the release of Cytochrome C from mitochondria and cleavage of Caspase cascades indicate the presence of turbulence as a result of apoptosis induction in Dis-treated cells. Moreover, NF-?B translocation was inhibited in Dis-treated cells. Our results indicate that Dis can target HCT-116 cells through the mitotic disruption and apoptosis induction.
Colorectal cancer (CRC) is the third leading cause of cancer-related mortality worldwide. Alarmingly, 700,000
deaths were reported for CRC incidence in 20161. It is expected that by 2030 the global rate of CRC will reach
more than 2.2 million new cases and 1.1 million deaths2. Increase in incidence and mortality of CRC is different
between developed and developing countries. Incidence and mortality rate of CRC in developed countries is
higher, but there is trend of increasing incidence in countries with low and middle incomes3. Like other types of
cancer cells, CRC cells have common hallmarks such as, uncontrollable growth, insensitivity to growth inhibitors,
resistance to apoptosis, indefinite replicative potential and their angiogenesis ability, which helps tumors to
survive and migrate to other parts of the body.
Besides surgery and radiotherapy, adjuvant chemotherapy drugs such as oxaliplatin and 5-fluorouracil (5-Fu)
are commonly used for the treatment of CRC4. Despite frequent use of these chemotherapy drugs, there have
been a large number of undesirable side effects observed during therapy, such as chest pain and cardiotoxicity5.
Moreover, treatment with these drugs can lead to failure due to resistance of cancer cells6. Therefore, new
therapeutic agents targeting different signaling pathways of cancer cells with lesser burden of side effects on normal
cells are much desired. Therefore, natural products are considered as potential candidates due to their low side
effects and high anti-cancer efficiency7.
Diosmetin (Dis) is a citrus flavonoid with anti-tumorigenesis properties against a variety of cancer cells
including hepatocarcinoma, leukemia, breast, lung and prostate cancer8. Dis is able to inhibit the proliferation
of cancer cells through different pathways. Although Dis inhibits polo-like kinase 1 (PLK1) as a progression
factor during mitosis, proliferation of different cancer cells such as A549, MDA-MB 468, LNCaP and PC3 cells
are inhibited in G0/G1 phase8?12. In prostate cancer, the arrest of cells occur as a consequence of reduction in
protein expression of cyclin D, cdk 2 and 4. Moreover, reduction in protein expression of Bcl-2 and c-Myc, whilst
overexpression of Bax, p27kip1 and Foxo3 encourages prostate cancer to progress to the apoptosis phase8. Dis
induces apoptosis not only in prostate cancer but also in leukemia cells by activation of extrinsic apoptosis
pathway and in hepatocarcinoma cells through the inhibition of NF-?B and activation of p5312?14. In addition, Dis was
identified as a metastasis inhibitor in hepatocellular carcinoma (HCC) cells via reserve effect of this compound
on matrix metalloproteinase MMP 2 and MMP 915. Although the cytotoxicity of Dis against Colo205, HT-29 and
Caco-2 colon cancer cells has been reported, the exact molecular mechanism of this compound in controlling
proliferation is yet to be elucidated. In the present study, we investigated the anti-colorectal cancer effect of Dis
against HCT-116 as one of the common human colorectal cancer cells. Besides, we introduce diosmetin (Dis) as a
potent polyphenol for combating CRC as an alternative therapeutic agent. In addition, the molecular mechanisms
involved and targeted signaling pathways were investigated at the gene and protein levels.
Cytotoxic effect of diosmetin against colon cancer cells. Cytotoxicity effect of Dis on HCT-116,
HT-29 colon cancer cells, and CCD-841 normal colon cells were assessed using MTT assay. The IC50 of Dis
against HCT-116 cells was 5.56 ? 0.48 ? g/ml, 3.58 ? 0.58 ? g/ml and 3.41 ? 0.64 ? g/ml after 24 h, 48 h and 72 h,
respectively. Moreover, the IC50 of Dis against HT-29 cells was 44.47 ? 5.31, 17.55 ? 2.96 and 11.46 ? 0.1,
respectively at the same time points. However, CCD-841 treated cells with Dis showed IC50 of 236.46 ? 9.99 ? g/ml,
51.95 ? 0.11 ? g/ml and 40.51 ? 9.99 ? g/ml, respectively at the same time points (Table?1). Among these three time
points the IC50 of Dis treated HCT-116 after 48 h was significantly lower compared to the IC50 of Dis after 24 h
while, IC50 of 48 h and 72 h was not significantly different. Therefore, the IC50 of 48 h was selected to investigate
the underlying molecular mechanism and signaling pathways of Dis. Moreover, cytotoxicity of Dis was compared
with 5-Fu as conventional anti-colon cancer drug (Table?1). Moreover, the IC50 of Dis against HT-29 was much
higher as compared to HCT-116 at the same time points, therefore HCT-116 colon cancer cells was selected for
further exploration of the mechanistic pathway of anti-cancer properties of Dis. Figure?1 shows the inhibitory
percentage of Dis and 5-Fu against HCT-116, HT-29 and CCD-841 cells.
Diosmetin activates intrinsic and extrinsic pathways of apoptosis. As shown in Fig.?2A, AO/PI
staining demonstrated that in the presence of Dis, morphology of HCT-116 cells was changed with the passing
of time. After 48 h exposure of the HCT-116 cells to Dis, cells started to have appearance of blebbing, which is a
sign for induction of apoptosis. After 72 h treatment, the numbers of dead cells increased. For Annexin-V assay,
Fig.?2B shows that apoptosis in treated HCT-116 cells with Dis was induced in time dependent manner. In the
control group, the population of cells in early apoptosis was increased from 4.15 ? 2.95% to 7.7 ? 0% after 72 h
of treatment. Late apoptosis was observed (1.2 ? 0.3% control; 18 ? 3.6% after 24 h; 26.3 ? 3.6% after 48 h; and
53.15 ? 4.35% after 72 h) after treatment of the cells with Dis at the different time points (Fig.?2C). The obtained
results demonstrated that Dis induced apoptosis in HCT-116 cells. In order to investigate which pathway of
apoptosis was involved, the activity level of caspase 8 (extrinsic apoptosis pathway), caspase 9 (intrinsic apoptosis
pathway) and caspase 3/7 (common in both apoptosis pathways) were measured using caspase cascade kit. As referred
in Fig.?2D, in the presence of Dis, cleavage of caspase 3/7, 8 and 9 were significantly increased in a time dependent
manner. These data shows that Dis instigates apoptosis through both the intrinsic and extrinsic pathways.
Intrinsic pathway. After evaluating the intrinsic and extrinsic apoptotic pathways, the features of each
pathway were assessed in detail for Dis treated cells. HCT-116 cells showed cell permeability aggravation and
loss of mitochondrial membrane potential when exposed to Dis as compared to the untreated cells. As shown
in Fig.?3, nucleus Hoechst 33342 (blue) is intact and uniform in untreated HCT-116 cells while treated cells
have fragmented nucleus. In the absence of Dis, YoYo dye (green) is not able to penetrate inside the cells since
the cell membrane is unified. Therefore, in control group intensity of green dye is low compared to the treated
cells. On the other hand, release of Cytochrome C (yellow) from mitochondria was observed in Dis-treated cells
but not in the untreated cells. In the treated group, reduction of Mitotracker dye (red) in the cytosol indicates
mitochondrial damage and increase in mitochondrial outer membrane permeabilization (MOMP) compared
to the control group. Moreover, gene expression results indicate that expression of bax which is a key factor in
Lower confidence Upper
limit confidence limit P-value
intrinsic apoptosis pathway, significantly increased in the presence of Dis16 (Table?2). The protein expression level
of Bax and Cytochrome C were accelerated while protein expression of Survivin (caspase 9 inhibitor) significantly
decreased in treated cells compared to the control. On the other hand, Hsp27 (Cytochrome C inhibitor)
expression significantly decreased in both gene and protein levels (Fig.?4 & Table?2).
Extrinsic pathway. In addition to activate Caspase 8, dead receptors such as TNFR and Fas showed increase
in their activities in the gene and protein levels. Consequently expression of FADD as a dead domain protein was
significantly increased (Fig.?4). Apart from the significant role of protein and genes which stimulated intrinsic and
extrinsic pathways individually, the high level protein expression of some other factors such as phosphorylation of
P53 plays a significant role on both pathways simultaneously in treated cells. Phosphorylation of P53 (s15 and s46)
can induce P21 which is known as a cell proliferation inhibitor17. As shown in Table?2, gene expression of setd2 as an
inducer of P53 significantly increased, while the expression of inhibitors of P53 (hdac1, 2, 5 and sin3) were decreased
significantly (P < 0.05)18?21. Protein expression of pro-caspase 3 was significantly reduced in Dis treated group as
compared to the control group (fold change = 0.75). On the other hand, protein expression of caspase 3 cleavage was
increased significantly up to 2.61 fold change in Dis treated group compared to the control group (Fig.?4).
Dis inhibits the translocation of NF?B. Although activation of TNF receptor can induce the extrinsic
pathway, it can also inhibit apoptosis through translocation of NF-?B from cytoplasm to nucleus. In order to
understand if Dis can inhibit NF-?B translocation, NF-?B assay was performed. As demonstrated in Fig.?5A and
B, following treatment of the cells with Dis, translocation of NF-?B was significantly reduced as compared to the
untreated group. Moreover, as shown in Fig.?5C Dis elevates the gene expression of NF-?B inhibitor (i?b-?)22.
Dis inhibits cell proliferation and arrests cells in G2/M. Flow cytometer was used to evaluate the effect
of Dis on cell arrest by measuring the DNA content of the cells in the presence of Dis at different time points as
compared to untreated cells. As shown in Fig.?6A,B, the first peak represents the DNA content of the cells in G0/
G1 and the second peak represents the DNA content of the cells in G2/M. Accumulation of the DNA content in
G2/M with time indicates that the cells were arrested during the mitotic division.
In order to identify the mechanism underlying mitotic arrest, signaling pathway involved in G2/M arrest
was investigated using Nanostring. As shown in Table?3, in the presence of Dis, expression of the genes that
are involved in mitosis division (ttk, pttg2, mad2l2, stmn1, smc3 and rad21) were significantly reduced23?31.
Meanwhile, gene expression of cyclin A and B inducer such as cdc25 was significantly decreased, expression of the
cyclin A and B inhibitors (p21, gadd45 and stratifin) were accelerated and consequently, gene expression of cyclin
A and B which are known to be cell regulators in G2/M, were inhibited32?39.
potential role of Dis in DNA synthesis. Proliferation of Dis-treated cells is not only influenced by mitosis
slippage, but also by inhibition of DNA replication. The results illustrated that the expression of genes, which are
involved in DNA repair (c19orf40 (faap24), ubb, nbn and msh2), DNA packaging (hist1h3h, hist1h3b, h3f3a and
h2a) and DNA replication (pole2, hells, pcna, mcm2, 4, 7, cdc7and prkar2b) were significantly decreased (Table?4).
Therefore, the obtained results suggest a potential role of Dis in inhibiting DNA synthesis in HCT-116 treated cells.
BMP is a potential mechanism involved in Dis triggered apoptosis. Nano string results indicate
that the gene expression of bmp8A, smad 9, id 1 and 2 in Dis-treated HCT-116 cells were significantly decreased
(p < 0.05) (Table?5). Gene bmp belongs to the tgf beta superfamily which stimulates smads including smad 9.
Activation of smad 9 can regulate the transcriptional factors such as id1 and 2 which are known as cell proliferative
factors and p21 inhibitors40?42. Therefore in the presence of Dis, apoptosis activation and cell cycle inhibition may
be triggered though the inhibition of TGF-? signaling pathway.
Apoptosis and cell proliferation inhibition are considered to be the main targets for chemotherapeutic drugs
in colon cancer treatments43,44. Besides the side effect of these drugs, treated tumors may develop resistance to
these chemotherapeutic agents and prolong the proliferation5. Contrary to chemotherapeutic drugs, anti-cancer
drugs from natural sources have shown lesser side effects. These limitations encourage new researches to focus on
developing more potent therapeutic drugs from natural sources45,46. Several natural products have been reported
to possess anti-tumorigenesis properties including quercetin, kaempferol, baicalein and apigenin7. Dis is a
flavonoid which is known for its anti-inflammatory and antioxidant properties10. Based on previous studies Dis
has anti-proliferative, anti-metastasis and pro-apoptotic effect on breast cancer and hepatocellular carcinoma
cells10,13,15. Moreover, cytotoxic effect of Dis against Colo205, HT-29 and Caco-2 cells demonstrates that Dis has
anti tumorigenesis properties against human colorectal cancer10,47,48. However the exact molecular mechanisms
underlying the anti-colon cancer effects of Dis are not well established. In the present study, we demonstrated that
Dis inhibits cell proliferation and activates apoptosis signaling pathways in human colorectal cancer. Moreover,
our study clearly demonstrates that activation of apoptosis and proliferation inhibition is likely to be intensified
through the inhibition of BMP pathway and NF-?B.
Our results indicate that Dis was cytotoxic for HCT-116 colon cancer cells (IC50 = 3.58 ? 0.58 ? g/ml)
however the cytotoxicity of this compound on non-cancerous colon cells was low at the same time point
(IC50 = 51.95 ? 0.11 ? g/ml). Therefore, Dis might be considered as a potent candidate to treat colon cancer. The
present study not only demonstrates the cytotoxicity of Dis against colon cancer but also shed a light on the
possible molecular signaling pathways of this compound against HCT-116 cells. Inhibitory effect of Dis against
HCT-116 was assessed in different time points. IC50 of Dis at 48 h (3.58 ? 0.58 ? g/ml) was significantly lower as
compared to the IC50 of this compound after 24 h (5.56 ? 0.48 ? g/ml). However, no significant difference was
observed between the IC50 of Dis at 48 h and 72 h (3.41 ? 0.64 ? g/ml). The IC50 of Dis after 48 h was slightly higher
than the IC50 of 5-Fu (3.07 ? 0.34 ?g/ml) as a registered drug at the same time point. Despite the similarity in the
IC50 doses of these two drugs, there was a significant difference between the IC50 of Dis (51.95 ? 0.11 ?g/ml) and
5-Fu (28.34 ? 0.67 ? g/ml) on normal colon cells CCD-841 after 48 h. Therefore, Dis might be a safer agent with
lesser cytotoxic effect on normal cells as compared to 5-Fu. Cytotoxicity properties of Dis in previous studies
demonstrates that Dis has the lowest effect on HCC (IC50 = 50 ?g/ml) followed by Colo205 (IC 50 = 29 ?g/ml) and
HepG-2 (IC50 = 12 ? g/ml) after 24 h treatment. Moreover, IC50 of Dis against non-cancerous cells such as
MCF10 (IC50 = 30 ?g/ml) and SK-Hep-1 (IC 50 = 100 ?g/ml) were higher compared to the cancer cells at the same time
point10,13,15,47. By comparing cytotoxicity results of HCT-116 cells with the other cell lines in previous studies, it
seems that HCT-116 cells (5.56 ? 0.48 ?g/ml) has more sensitivity to Dis compared to the other types of cell lines
except MDA-MB 468 (IC50 = 1.35 ? g/ml) after 24 h treatment. Therefore it can be concluded that Dis has potent
anti-tumor activity with less cytotoxic effect on normal cells. The possible reason for the extreme difference in the
dosage of Dis towards colon cancer cells and normal colon cells, may lie in stimulation of apoptosis pathways by
Dis and interference with mitotic progression in cancer cells.
Eternal life of cancer cells occurs when their apoptotic signaling pathways are not activated. Apoptotic agents
such as Dis can promote the destiny of cancer cells towards death by activating apoptotic factors such as caspase
cascades. Activation of Caspase 8 and 9 occurs as a result of activation of the extrinsic and intrinsic apoptosis
pathway, respectively. Meanwhile activation of Caspase 3/7 is involved in both apoptotic pathways. As shown
in Fig.?7, activation of intrinsic apoptotic pathway is dependent on the stimuli from inside the cells. Apoptotic
mediators accumulate in the intermediate space between the two membranes of mitochondria. Therefore,
pro-apoptotic factors such as Bax increase the permeability of mitochondria through mitochondria outer
membrane permeabilization (MOMP) process. As a result, the release of apoptotic mediators such as Cytochrome C
combines with pro-Caspase 9 to form a complex named apoptosome. When pro-Caspase 9 cleaves to Capspase
9 it activates its downstream caspase cascades such as Caspase 3 and 7. Inhibitor proteins such as Survivin which
is a member of inhibitors of apoptosis proteins (IAP) can inhibit the activation of Caspase 9 and 7 by blocking
their active sites16.
In the present study, activation of Caspase 9 in Dis-treated cells together with increase in mitochondrial
membrane permeability, release of Cytochrome C and Bax indicate that Dis can induce the intrinsic apoptotic pathway.
Moreover, in the presence of Dis, protein expression of IAPs such as Survivin and Hsp27 (Cytochrome C
inhibitor) is reduced in cancer cells.
In addition to intrinsic apoptosis pathway, extrinsic apoptosis pathway is triggered by death factors such as Fas
and TNF-?. Caspase 8 acts as a key factor in this pathway16. In our study, HCT-116 treated cells with Dis increase
the Fas and TNF receptor at both gene and protein levels. When the complex of death ligands and receptors in
Dis-treated cells increase, configuration of the receptors will change and adaptor proteins such as FADD bind to
the cytoplasmic end of the receptors. Increase in the expression level of FADD in treated cells cause the activation
of Caspase 8 which initiates a cascade of Caspase activation such as Caspase 3 and 716. Rapid ignition of apoptosis
in Dis-treated cells, is partially related to the activation of P53 protein (Fig.?7). P53 is one of the most important
tumor suppressors, which is altered in most human cancer cells. P53 is able to induce apoptosis by up regulation
of death receptors such as Fas and pro-apoptotic Bcl-2 family member Bax49,50. Our gene expression results
illustrated that expression of setd2 as an inducer of p53 was increased whereas expression level of p53 inhibitors such
as hdac1,2,5 and sin3 was decreased18?21. Although gene expression of p53 inducers and inhibitors were altered in
Dis treated cells, gene expression of p53 remains unchanged. Our protein array results illustrate that activation of
P53 occurred as a results of phosphorylation of P53 in ser15, ser46 and ser39251,52.
NF-?B is a transcription factor, which is a key mediator in inflammatory response. NF-?B is activated through
inflammatory factors such as TNF-?. Besides the inflammation, NF-?B has other downstream effectors that
contribute to tumorigenesis and apoptosis inhibition. In general, translocation of NF-?B from cytoplasm to the
nucleus causes apoptosis inhibition via activation of IAPs such as Survivin. Moreover, NF-?B can be accumulated
in cytoplasm by NF-?B inhibitor (i?b-?)22,53. In Dis-treated cells, TNF-? receptors were increased, which in turn
activates the extrinsic apoptosis pathway. As described previously, TNF-? is an NF-?B inducer. In our study,
despite elevation of TNF-? receptors in treated cells with Dis, translocation of NF-?B was inhibited via
overexpression of i?b-?. Moreover, reduction in protein expression of Survivin confirms NF-?B inhibition. Hence, Dis
activates the apoptosis both through the engagement of apoptotic factors and inhibition of NF-?B (Fig.?7).
As described before, previous studies claimed that Dis has anti-proliferative properties by arresting MDA-MB
468 breast cancer cells in G1 phase10. By comparing these data with ours, it can be concluded that the molecular
mechanism of Dis seems to vary in different type of cells.
Proliferation rate in cancer cells are higher as compared to the normal cells. For this matter, DNA replication
needs to be accelerated. This feature makes cancer cells more susceptible to chemotherapeutics drugs which
targets DNA replication and repair61.
The mcm2, 4 and 7 are involved in forming replication fork and recruitment of other DNA replication related
elements. They are also responsible for activation of Helicase which unwinds dsDNA to generate ssDNA templates
for DNA polymerase function62,63. Subunits of msms are mainly induced through cdc7 and prkar2b genes64,65.
While DNA is unwound, DNA polymerase is initiated DNA replication. During the replication of DNA, DNA pol
? extend the leading strand and pcna accelerates the DNA synthesis. DNA pol ? is not only involved in replication,
but it has role in DNA repair66,67. The function of pcna is inhibited by p2168. In the presence of Dis, gene
expression of mcm2,4,7, hells (Helicase), cdc7, prkar2b, pole2 (DNA pol ?) and pcna were inhibited while, p21 expression
was elevated in HCT-116 cells (Fig.?8A).
During and after DNA replication, based on the type of error, DNA repair system initiates repair of DNA. Any
disruption in DNA repair system, causes accumulation of damaged DNA. Since DNA repair system is defective
in cancer cells, finding a way to turn dysregulated repair system against themselves and induce tumor death, is a
goal for DNA repair inhibitor agents69. In our study, genes such as msh2, c19orf40 (faap24), nbn and ubb, which
are involved in different types of DNA repair system were inhibited in Dis-treated cells (Fig.?8B)70?74.
Beside the inhibition of genes which are involved in DNA synthesis and DNA repair by intermediation of Dis,
expression of those genes which are involved in forming nucleosome such as hist1h3h, hist1h3b(h3), h3f3a and
h2a were decreased75. In order to construct chromosomes, DNA needs to condense through different form of
packaging. Formation of nucleosome is the basic unit for chromosome construction in which segment of DNA
wound around eight histone proteins including h3 and h2a75,76. As described, Dis is able to inhibit HCT-116
proliferation not only through mitotic slippage, but also by the interruption of DNA synthesis system, inhibition of
DNA repair system and delay in forming nucleosome (Fig.?8A).
Our data indicates the anti-cancer properties of Dis against HCT-116 cells. Mechanism of action of Dis against
HCT-116 might be through activation of apoptosis, mitotic slippage and NF-?B inhibition. Besides, the other
potential anti-tumorigenesis mechanism of Dis might be through inhibition of DNA synthesis/ repair system and
BMP pathways. Our data support previous studies for apoptotic properties of Dis through activation of P53, Bax
and inhibition of NF-?B. However, in spite of prostate cancer and breast cancer, cell cycle proliferation inhibited in
G2/M phase for HCT-116 cells through mitotic slippage and inhibition of cylin A/B rather than in G0/G1 phase8?14.
Despite previous results which claim that Dis inhibits TGF-? and Smad 3 in HepG-2, no significant
difference were observed in HCT-116 treated cells13. However, our results indicate that Dis inhibit bmp and smad 9 in
HCT-116 cells. Moreover, our results did not support previous findings based on inhibition of mmp 2 and mmp
9 in HCC cells15. These differences indicate that mechanism of action of Dis might vary between different types
of cancer cells.
In conclusion, our findings demonstrate that Dis has anti-cancer properties against HCT-116 cells and less
cytotoxic effect against non-cancerous colon cells. Our study claims that the mechanism of action of Dis against
HCT-116 cancer cells might be through the induction of apoptosis and inhibition of cell proliferation. Dis is
able to induce apoptosis mediated by the mitochondria and apoptosis mediated by membrane death receptor.
Dis activates the intrinsic apoptotic pathway through a mechanism in which Dis induces Bax to form a channel
in the outer membrane of mitochondria and releases Cytochrome C to activate Caspase 9, whereas Caspase 8 is
activated when dead domain of TNF-?/Fas cleaves pro-Caspase 8. Dis also accelerates apoptosis when NF-?B
arrested by I?B and hence transcription of anti-apoptotic factors is inhibited. Induction of pro-apoptosis
component in the presence of Dis on one hand and inhibition of anti-apoptosis on the other, accelerate apoptosis trend
in HCT-116 cells.
Dis arrests HCT-116 cells in G2/M phase either through inhibition of cyclin A/B or mitotic slippage. Activation
of cyclin A/B inhibitors (p21, gadd45, stratifin) and prevention of their inducer (cdc25) play an important role in
the down regulation of cyclin A/B in Dis-treated cells. Moreover, in the presence of Dis, mitosis is disrupted in
HCT-116 cells due to early detachment of sister chromatids and interruption in microtubule dynamic. In
addition, by inhibition of bmp, cell cycle arrest is accelerated due to activation of p21. As BMP and DNA synthesis/
repair inhibition are considered as potential pathways that are affected in Dis-treated cells, which need further
investigations to explore the exact mechanisms by which diosmetin affected these pathways.
Compound and cell lines. Diosmetin was purchased from Abcam (Cambridge, UK) with the molecular
weight of 300.26 g/mol and purity >98%. 5-Fluorouracil (5-FU) was obtained from MP biomedical, lllkirch,
France. HCT-116, HT-29 (human colon cancer) and CCD-841 (human non-cancerous) cell lines were obtained
from ATCC, USA. All assays were performed in three independent experiments and the results are presented as
mean ? SD of triplicates.
Cell culture. HCT-116, HT-29 (colon cancer) and CCD-841 (non-cancerous) cell lines were obtained from
ATCC (VA, USA). Cells were cultured in DMEM medium (HyClone, Logan, Utah, USA) supplemented with 10%
FBS and 1% penicillin and streptomycin (Biowest, France). Cells were incubated in incubator at 37 ?C with 5%
CO2. Negative control for all assays was represented by 0.02% DMSO (vehicle) in untreated medium.
Cytotoxicity assay. Cytotoxicity property of Dis against HCT-116 and CCD-841 cells were measured by
MTT assay as described by Mossman77. Briefly, cells were treated with different concentration of Dis and 5-Fu
(positive control) with serial dilution factor of 2 for 8 concentrations in 96 well-plate for different time points
(24 h, 48 h and 72 h). Subsequently at the end of each time point 20 ? l of MTT (5 mg/ml, Affymetrix,Cleveland,
OH, USA) were added to each well followed by 4 h incubation. The solutions were aspirated after 4h and 100 ? l
of DMSO were added to each well. Absorbance for each well was read at 570 nm as measured by using microplate
reader (Tecan Infinite multimode, M?nnedorf, Switzerland). Cytotoxicity (cell death) was measured by using the
Cell death (%) =
sample OD ? control OD
This assay was performed in triplicates in three independent experiments. The cytotoxicity property of Dis
was presented as IC50 value.
Acridine orange/propidium iodide (Ao/pI) double staining assay. Morphological changes of HCT
116 cells in the absence and presence of Dis were assessed by AO/PI (Santa cruz, CA, USA) double staining assay
as previously described78. In brief, cells were seeded in 25 cm2 culture flask followed by treatment with Dis at
different time points of 24 h, 48 h and 72 h. Cells were then harvested and washed with PBS. Pellets were stained
with 10 ? l of AO/PI. Morphological changes of the cells were detected using the fluorescence-inverted microscope
(Nikon, Japan) in which green color represents healthy cells while dead cells were represented by red color.
Annexin-V-FITC assay. Inductions of early and late stages of apoptosis by Dis were investigated using
Annexin-V-FITC assay according to the manufacturer?s instructions (BD Biosciences, San Jose, CA, USA).
Briefly, 106 HCT-116 cells were seeded in 25 cm2 flask followed by treatment with Dis for 24 h, 48 h and 72 h.
Treated cells were harvested and washed twice with cold PBS and suspended in binding buffer followed by
transferred of 105 cells to the 6 ml polystyrene round-bottom FACS tubes. Cells were incubated with 5 ?l of PI and 5 ?l
of Annexin-V-FITC and introduced to the FACSCanto?II instrument (BD, San Jose, CA, USA).
Bioluminescent assays for caspase 8, 9 and 3/7 activities. To assess the effect of Dis on caspase
cascades in treated cells, activities of caspase-Glo 3/7, caspase-Glo 8 and caspase-Glo 9 assays (Promega, Madison,
WI, USA) were performed. Briefly, HCT-116 cells with the density of 104 cells/well were seeded in 96 well-plates.
Cells were treated with Dis for 24 h, 48 h and 72 h. This assay was performed in triplicates for each time point. A
total of 6 wells were preserved for samples without cells but with media and reagents as the blank samples, and
for the untreated cells as negative control. At the end of incubation time, 100 ? l caspase-glo mixture were added
to each well and incubated for 1 h in the dark according to the manufacturer?s instructions. Optical density was
measured via multimode microplate reader (Tecan Infinite, M?nnedorf, Switzerland).
Multiple cytotoxicity assay. Cellomics multiparameter cytotoxicity 3 kit (Thermo Scientific?, Pittsburgh,
PA, USA) was used to investigate the vital apoptotic events in HCT-116 cells in the presence of Dis. Cytotoxicity
3 assay was used to provide information about the changes in nuclear morphology, cell membrane integrity,
cytochrome C release and mitochondrial outer membrane permeabilization (MOMP). HCT-116 cells were
seeded in 24 well-plate and covered by a cover slide followed by treatment with Dis for 24 h, 48 h and 72 h. Treated
cells were stained with YoYo dye (life technology, CA, USA) and Mitotracker dye (life technology, CA, USA)
followed by fixation and blocking procedures according to the manufacture?s protocol. Primary cytochrome
C (Thermo Scientific?, Pittsburgh, PA, USA) antibody and secondary DyLight 650 (Thermo Scientific?,
Pittsburgh, PA, USA) were added to each well and incubated for 1 h in the dark. Hoechst 33342 dye (Thermo
Scientific?, Pittsburgh, PA, USA) was added in the last step to stain the nuclei of the cells. Cells were washed
and cover slides were transferred on the slide and introduced to confocal Leica TCS SP5 II microscope (Leica
Microsystems, Mannheim, Germany). Intensity of each dye was measured by using LAS X software.
Translocation of NF-?B. Confocal microscope was used to detect the suppressive effect of Dis on
translocation of NF-?B from cytoplasm to nucleus induced by TNF-? (Santa Cruz, CA, USA) using cellomics nucleus
factor-?B (NF-?B) activation kit (Thermo Scientific?, Pittsburgh, PA, USA). HCT-116 cells were seeded
and treated with Dis at different time points in 24 well-plate covered with cover slide. An amount of 150 ng/
ml of TNF-? was used as a stimulator for 24 h. Cells were then fixed and stained according to the
manufacturer?s instructions. Translocation of NF-?B was investigated via confocal Leica TCS SP5 II microscope (Leica
Microsystems, Mannheim, Germany) and intensity of NF-?B was measured by using LAS X software.
Cell cycle. Flow cytometry analysis was performed to determine any disruption during the cell cycle of
HCT116 cells in the presence of Dis. HCT-116at density of 2 ? 106 cells were seeded and treated for 24 h, 48 h and
72 h in a 75 cm2 flask. Negative control was treated with 0.02% DMSO. After fixation of the cells with 70%
ethanol, cells were preserved in ?20 ?C overnight followed by washing and staining with 500 ? l of PI/Rnase dye
(BD Biosciences, San Jose, CA, USA) and incubated for 30 min. DNA content analysis was performed using BD
FACSCanto II flow cytometer instrument and BD FACS Diva software (BD FACSCanto?II, San Jose, CA, USA).
A total of 15,000 events per sample were recorded for analysis. ModFit LT version 3.0 software was used for
analyzing the data.
Gene expression profiling. Gene expressions of the cancer canonical pathways were measured by
NanoString nCounter. HCT-116 cells were treated with Dis for 48 h and total RNA were extracted by using
RNeasy Mini Kit (QiAgen, Hilden, Germany) according to the manufacture?s protocol. Concentrations of RNA
were checked through NanoDrop 2000C (Thermo Scientific?, Pittsburgh, PA, USA). Pancancer pathways panel
on Nanostring nCounter system (Nanostring Technologies, Inc., Seattle, WA, USA) were used to investigate the
gene expression profile of cancer pathways. A total of 770 genes were investigated and compared between Dis
treated and untreated cells. The list of investigated genes is provided as supplementary data (Table?S1). RNA
was hybridized, purified and immobilized before being aligned on the nCounter cartridge and introduced to
Digital Analyzer was utilized for data collection according to the manufacturer?s instruction. Briefly, RNA
samples were added to the master mix contain hybridization buffer and reporter CodeSet. Capture ProbeSet was
added to this mixture and introduced to pre-heated 65 ?C thermal cycler for 16 h incubation. After removing
the unbound probe, target/probe were immobilized and aligned on the nCounter Cartridge. nCounter digital
analyzer was used to collect the nCounter cartridge data and data were analyzed using nSolver 3.0 analysis
software. Significant genes were choose based on the false discovery rate (FDR) <0.05 and log2 fold change ? ? 0.5
supplementary data (Figure?S1).
protein array. Apoptosis protein expression profiling was performed using human apoptosis array (R&D
Systems, Minneapolis, MN, USA) according to the manufacturer?s protocol. In brief, 2 ? 106 HCT-116 cells were
seeded and treated with Dis in 75 cm2 flask and incubated for 48 h. Cells were harvested and lysed via lysis buffer
reagent and was quantified using Pierce BCA protein assay kit (Thermo Scientific?, Pittsburgh, PA, USA). The
membrane provided was blocked in blocking solution reagent and treated overnight with cell lysates protein. The
membrane was washed and subsequently incubated with detection antibody cocktail and streptavidin
horseradish peroxidase-conjugated. Chemiluminescent reagents were added to the membrane and then exposed to X-ray
film to detect the protein expression levels in treated cells as compared to untreated cells. Images were quantified
via ImageJ software (NIH, USA). The list of investigated proteins is provided as supplementary data (Table?S2).
statistical analysis. All the data were expressed as mean ? standard deviation (SD) of three replicates.
Statistical analyses were performed using one-way analysis of variance (ANOVA) with Tukey?s multiple
comparisons. Data were analyzed with graph pad prism, version 6.07 for windows. The values of p < 0.05 were considered
List of investigated genes and proteins are available in supplementary data. Gene expression of investigated genes
are presented in volcano plot in supplementary data.
The authors would like to thank the University of Malaya, Malaysia, for supporting this study through IPPP grant
M.A.A., Z.M., and S.K. designed the study; S.K. carried out the experiments; M.A.A., Z.M., A.S., and S.K.
analyzed the data; M.A.A., Z.M., A.S., and S.K. interpreted the findings; S.K. drafted the manuscript and all
authors reviewed the manuscript and approved the final version for publication.
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-41685-1.
Competing Interests: The authors declare no competing interests.
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