Dentatin triggers ROS-mediated apoptosis, G0/G1 cell cycle arrest and release of Th1-related cytokines in colorectal carcinoma cells

Naturally occurring carbazole alkaloid, Dentatin (DTN), is known to inhibit human cancer cell lines. However, it’s inhibitory and immunomodulatory activities in colorectal carcinoma HCT-116 cells remain obscure. This study compares DTN’s inhibitory effect against 5-FU in HCT-116 cells using in vitro anticancer assays including cell cytotoxicity, migration, colony formation, cell cycle and apoptosis, while assessing its immunomodulatory activity via Th1/Th2/Th17 inflammatory cytokines assay. DTN triggered the intrinsic and extrinsic apoptosis pathways by actuating caspases-8, -9 and -3 while inhibiting cell migration and colonies formation, and inducing cell cycle arrest at the G0/G1 phase. Accumulation of iROS with loss of MMP and DNA damage was observed with elevation of several Th1 cytokines, the TNF-α and IFN-γ with lower pro-inflammatory IL-6 compared to 5-fluorouracil. In conclusion, DTN triggered ROS-mediated apoptosis with release of inflammatory cytokines, potentially evoking anticancer immunity in CRC. Further validation of DTN’s mechanism in vivo is warranted.


Introduction
Colorectal cancer (CRC) develops regardless of chromosomal instability sequence or serrated neoplasia pathways are deemed as a major predicament in the sphere of oncology.Though armed with unprecedented technological advancements, contemporary medical science howbeit still fails to put this disease at ease.Nearly two million people have succumbed to this malady, with a considerable number of deaths ( ∼ 935,000) reported in 2020 [1].In Malaysia, CRC cases are ethnicity dependent.Such notion was validated as CRC incidences per 100,000 were reported to be higher in Chinese (27.35) compared to Malay (18.95), and Indian (17.55) ethnicities, though the exact reason is unknown, this paradox is believed to be influenced by genetic predisposition and diet [2].CRC was initially considered as an "old age" disease due to its proclivity to be diagnosed in older individuals, not until recent ubiquitous diagnoses were made in younger individuals ( < 50 years).This so-called early-onset colorectal cancer (EOCRC) has risen at a very alarming rate, which is expected to increase by > 140% by 2030.A study showed that Malaysian EOCRC patients were usually diagnosed at an advanced stage and associated with more aggressive tumour characteristics, which is also parallelly observed globally [3,4].
Treatment of CRC is majorly comprised of surgery and chemotherapy.Advanced CRC patients are put under treatment via intravenous injection of 5-fluorouracil (5-FU), while curative surgery is merely limited for patients with early stages and resectable tumours.Since its approval, 5-FU remains as the utmost palliative regimen for treating CRC, however, notoriously incriminated with insurmountable side effects, fatal toxicities and resistance [5,6].Hence, researchers are much obliged to discover natural compounds (NCs) from plants that have the possession of potent anticancer activities with minimum toxicity.Camptothecin and Taxol (paclitaxel) isolated from Camptotheca acuminate and Taxus brevifolia, respectively, were approved by the Food and Drug Administration (FDA) and used singly to treat several cancers [7].These NCs interfere with the initiation, development and progression of cancer cells via modulation of various mechanisms including cellular proliferation, differentiation, apoptosis, angiogenesis and metastasis [7].
Additionally, some NCs were reported to have immunomodulatory activity by modulating immune responses against cancer.Amongst these responses, the release of danger-associated molecular patterns (DAMPs) from dying cells to the tumour microenvironment (TME) could trigger immunogenic cell death (ICD), a type of cell demise that is associated with longterm anticancer immunity that deemed as the most important anticancer strategy [8].Some inflammatory cytokines such as IFN-γ and TNF-α are considered as DAMPs as they could evoke the activation of ICD.While some other cytokines such as IL-6 and IL-17A might involve in other types of inflammation, such as induction of tissue damage and inauguration drug resistance [9].Thus, to probe into cytokines that are released during cell demise, triggered by anticancer drugs into the extracellular milieu is vital in defining its role in the immune response against cancers.
Dentatin (DTN), a carbazole alkaloid that can be isolated from the plant Clausena excavata, is native to the Southeast Asian locale.It has been discovered to exert anti-proliferative effects on breast, prostate and liver cancer cells concomitantly being relatively non-toxic on normal cells and animal models [10][11][12][13].DTN induced predominantly reactive oxygen species (ROS) mediated apoptotic cell death by way of modulation of apoptotic molecules and subsequent activation of both intrinsic and extrinsic pathways in colorectal adenocarcinoma, HT-29 cells [14].However, its underlying mechanism on colorectal carcinoma HCT-116 cells remains obscure.In CRC drug discovery, both cell panels were typically used in tandem to discover the preclinical susceptibility of a drug against CRC.This is due to their complementary differences such as gender and mutation.HT-29 cells have mutated p53 gene, while HCT-116 cells possessed mutated PI3KCA and K-RAS genes.All these mutations are responsible for CRC-mediated drug resistance which highly predetermined the patient's proneness towards certain chemotherapeutic drugs [15].
In this study, several in vitro assays were executed to determine the inhibitory effect of DTN on HCT-116 cells while assessing its impact on the secretion of inflammatory cytokines during cell demise against CRC cells compared to 5-FU.

Cell culture
Colorectal carcinoma, HCT-116 (ATCC R CCL-247 TM ) and human primary colon, HPC (T4056, ABM) cell lines were purchased from American Type Culture Collection (Rockville, MD, USA) and Applied Biological Materials Inc. (ABM, Richmond, Canada), respectively.Roswell Park Memorial Institute (RPMI) 1640 (Sigma) supplemented with 10% of foetal bovine serum (Gibco), 2 mM L-glutamine, 100 units/ml of penicillin and 100 μg/ml of streptomycin (all acquired from Life Technologies, Australia) were used to culture both cells in a 37°C humidified incubator with 5% CO 2 .In all of the analysis, passage number was maintained to be around 5-15 passages.

Cell viability assay
Cell viability assay was performed to determine the IC 50 values (50% cell growth inhibitory concentrations) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2Htetrazolium bromide (MTT) assay as previously described [16].Briefly, HCT-116 and HPC cells plated (2 × 10 5 cells/well) in 96-well plates were subjected to treatment with DTN and 5-FU (concentration ranging from 0 to 20 μg/ml) for 24 and 48 h.MTT reagent (5 mg/ml) was introduced to each well before it was further incubated in a dark condition for 4 h.Absorbance was read using a microplate reader at OD570 nm following 10 min of incubation with DMSO.

Morphological observation
HCT-116 cells (5 × 10 3 cells/well) were plated in 96-well plates and treated with DTN with concentrations ranging from 0 to 20 μg/ ml for 24 and 48 h, respectively, following which the cell morphological changes were observed and captured under a phase-contrast microscope.

Clonogenic assay
Colonies formation was assessed using the clonogenic assay.Briefly, suspension of HCT-116 cells (500 cells/well) was seeded in six-well plates, followed by treatment with IC 50 concentration of DTN and 5-FU for 48 h at 37°C humidified incubator with 5% CO 2 .Upon incubation, the culture media was replaced with fresh media and allowed to grow for seven days prior to staining with crystal violet solution (0.5% w/v).The stained colonies were counted using densitometric software and a single colony was captured under an inverted microscope.

Cell migration assay
The cell migratory impact of HCT-116 cells was measured via the wound healing assay.HCT-116 cells with the density of 5 × 10 5 cells/well were seeded in six-well plates and mechanically scratched with a 10 μl pipette tip, following which the cells were treated for 48 h with IC 50 concentration of DTN and 5-FU, respectively.The wound gap was photographed using a phase-contrast inverted microscope (Leica, Germany) and the graphic was analysed and measured via LAS application suite X v3.8 software (Leica, Germany), at 0 and 48 h of treatments.

Apoptosis assay
The apoptotic cells were acquired using the FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen TM ) according to the manufacturer's instruction.In brief, HCT-116 cells (1 × 10 5 cells/well) were let confluence in six-well plate and were treated with IC 50 concentrations of DTN and 5-FU for 48 h.Cells were harvested, washed with phosphate-buffered saline (PBS) and suspended in binding buffer at the concentration of 1 × 10 6 cells/ml.The solution was incubated with 5 μl of fluorescein isothiocyanate (FITC) Annexin V and 5 μl propidium iodide (PI) and incubated in a dark condition for 15 min at room temperature.The sample was then quantified using a FACS Canton 11 cytometer (Becton-Dickinson, USA) and analysed by the Cell Quest software.

Intracellular reactive oxygen species
Intracellular ROS (iROS) was measured using ROS-ID R Total ROS (Enzo Life Sciences) detection kit according to the manufacturer's instruction.HCT-116 cells (1 × 10 3 cells/well) were seeded in 96-well plates and treated with IC 50 concentration of DTN and 5-FU for 48 h.Following that, the cells were treated with ROS detection solution and measured using a fluorescence microplate reader with fluorescein (excitation/emission: OD490/525 nm).

Measurement of mitochondrial membrane potential (MMP)
Mitochondrial membrane potential (MMP) was measured using JC-1 Mitochondrial Membrane Potential Detection Kit (Biotum, CA, USA) according to the manufacturer's instruction.Briefly, HCT-116 cells with the density of 1 × 10 3 cells/well were plated in 96-well plates and treated with the effective concentration of DTN and 5-FU for 48 h before the media was removed and replaced with JC-1 reagent and incubated at 37°C in a cell incubator for 15 min.Later, JC-1 reagent was removed from individual wells and replaced with PBS.The plates were measured using a fluorescence microplate reader; red fluorescence (excitation: OD550 nm, emission: OD600 nm) and green fluorescence (excitation: OD485 nm, emission: OD535 nm).

DNA damage assay
Cell DNA damage was observed via gel electrophoresis.HCT-116 cells (1 × 10 5 cells/well) seeded in six-well plate for a day were treated with the effective concentration of DTN and 5-FU for 48 h.Then, the DNA was extracted using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.
In brief, cells were detached using trypsin, washed with PBS and lysed via the addition of 500 μl of lysis buffer (10 mM Tris-HCl, pH 7.8; 5 mM EDTA and 0.5% sodium dodecyl sulphate) containing 50 μg/ml proteinase K and incubated at 60°C for 3 h.The resulting DNA was resuspended in a wash buffer and filtered through a spin column tube.Finally, the DNA was eluted in the elution buffer, stained and analysed on 1.5% agarose gel electrophoresis.

Caspases 3, 8 and 9 activities
Caspases 3, 8 and 9 were assayed using the caspase kits from Elabscience Biotechnology Co., Ltd., (Texas, USA) with catalogues number E-CK-A311, E-CK-A312 and E-CK-A313, respectively.Succinctly, HCT-116 cells (1 × 10 5 cells/well) were seeded in six-well plate for a day and treated with IC 50 concentration of DTN and 5-FU for 48 h, HCT-116 cells were lysed using lysis buffer on ice and subjected to mixing with colourimetric substrates of the respective caspases.The samples were incubated at 37°C for 4 h before the OD value was acquired using a microplate reader.Caspases activity was reported in fold per control/untreated.

Cell cycle analysis
The cell cycle analysis was performed using BD bromodeoxyuridine (BrdU) FITC Assay according to the manufacturer's instructions (BrdU Flow Kit; BD Pharmingen, San Diego, CA, USA).Briefly, cancer cells were seeded (1 × 10 5 cells/well) in six-well plate.On the next day, cells were treated with IC 50 concentration of DTN and 5-FU for 48 h.Then, 10 μM BrdU was added to each well and incubated for 30 min.Following trypsinization, the cells were further incubated with 20 μl anti-BrdU-FITC for 20 min as well as with 2.5 μl of 7-amino-actinomycin D (7-AAD) for 15 min.Further analysis using the FACS-Canton 11 has been performed following 10,000 events (Becton-Dickinson, San Diego, CA, USA).

Cytokine assay
Multiple cytokines were simultaneously determined using the Human Th1/Th2/Th17 CBA kit (BD Biosciences, San Jose, CA, USA).Briefly, 50 μL of the treated cell's supernatant was collected and diluted in assay diluent (1:2 v/v) provided by the manufacturer.This process was preceded by mixing the dilution with specific cytokines antibodies (IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ , IL-17A) and phycoerythrin-conjugated detection antibody.The sample was then incubated for 3 h in the dark condition before it was washed with wash buffer and centrifuged at 200 g for 5 min.The resulting pellet was resuspended in 300 μl of wash buffer and subjected to flow cytometry analysis using the BD FACS Canto II Flow Cytometer.The acquired data were further deciphered via FCAP Array TM Software (BD Bioscience) and cytokines concentration was reported in pg/mL [17].

DTN prominently inhibited HCT-116 cell's viability compared to 5-FU
MTT assay was used to assess the effect of DTN and 5-FU on the viability of HCT-116 and HPC cells.DTN was found to inhibit HCT-116 cell viability with dose and time-dependent manners (Figure 1

DTN triggered apoptotic-related morphological changes of HCT-116 cells
Under the phase-contrast inverted microscope, HCT-116 cells treated with DTN (0-20 μg/ml) for 24 and 48 h  showed that it caused morphological changes related to apoptosis.Photographs depicted treated cells underwent clear signs of cytoplasmic shrinkage and membrane blebbing (Figure 2).

DTN inhibited HCT-116 cells migration and colonies formation
Wound healing assay performed on HCT-116 treated with IC 50 concentrations of DTN (1.90 μg/ml) and 5-FU (2.61 μg/ml) for 48 h showed visible observation of inhibition of cells migration via retardation of the ability to close the wound-gap area was detected in both treated compounds (Figure 3(A,B)).Similarly, colony formation assay revealed that both compounds also inhibited colony formation of HCT116 cells.The control group exhibited mean of colony count of 645.3 ± 31.0 colonies, which profound diminution of colonies was measured in DTN (5.3 ± 2.5 colonies) and 5-FU (5.6 ± 3.1 colonies) treated cells (Figure 3(C,D)).These findings suggest similar to 5-FU, DTN also inhibits metastasis and reproductive viability of HCT-116 cells.

DTN accumulates iROS, and triggers deprivation of MMP and damage of DNA
Accumulation of ROS intracellularly might alter the mitochondrial metabolism that often presented with loss of MMP and upregulation of caspase 9 which is preceded by cell demise with DNA damage.Treatment of HCT-116 cells with DTN (1.90 μg/ml) compared to 5-FU (2.61 μg/ml) at 48 h were subjected to ROS, MMP and DNA fragmentation assays.Strikingly, the result revealed that DTN triggered ROS in HCT-116 cells, which was two-fold higher than 5-FU (Figure 4(H)).This finding was preceded by loss of MMP whereby DTN and 5-FU triggered significant deprivation of MMP compared to control (Figure 4(I)).The DNA fragmentation assay revealed fragmented DNA indicating the occurrence of DNA insults (Figure 4(J)).

DTN actuated G0/G1 mediated cell cycle arrest
Apoptosis is usually presented with cell cycle arrest.Flow cytometry plot of BrdU FITC dye incubated with HCT-116 cells treated with DTN (1.90 μg/ml) and 5-FU (2.61 μg/ml) for 48 h revealed that DTN impeded cell cycle progression of HCT-116 cells prominently at the G1/G0 phase (Figure 5).Meanwhile, as previously known cell cycle arrested by 5-FU prominently involved the S phase.

DTN sets off Th1 related inflammatory cytokines release in HCT-116 cells
Inflammatory cytokines are one the prominent entities for activation of immunogenic cell death and mediators for broad inflammatory processes.We further sought to explore the ability of DTN to actuate the secretion of inflammatory cytokines in HCT-116 cells compared to 5-FU.The supernatant of cells treated with DTN (1.90 μg/ml) and 5-FU (2.61 μg/ml) for 48 h were incubated with cytokines-specific antibodies (IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and IL-17A) and measured via flow cytometry.Compared to control, significant increment of cytokines IL-2, IL-6, IL-10, TNF-α, IFN-γ and IL-17A were observed in cells treated with both compounds.When compared to treatment with 5-FU, there is no significant difference observed in the level of cytokines IL-2, IL-4 and IL-10 secreted by the DTN-treated cells.However, DTN triggered a significant elevation of IFN-γ and TNF-α.Inversely, although IL-6 was also significantly increased in DTN-treated cells' supernatant compared to control, it was three-fold lower than the 5-FU treated cells.5-FU showed an excessive increment of IL-6 compared to control.While IL-17A was significantly secreted higher in 5-FU treated cells compared to DTN (Figure 6(A-H)).

Discussion
The CRC cases are plummeting in developed countries, to a great extent owing to the availability of routine screening programmes and vast advocacy of awareness.However, the reality remains opposite in developing countries [18].The administrations of intravenous 5-FU-based regimens are the underpinning  treatment in CRC chemotherapy.Although they are effective, most patients were persistently inflicted with resistance, relapse, and fatal setbacks.The possession of lower side effects, cost-effectiveness and ease of accessibility are the prominent facets of a good anticancer drug, which are mainly embodied by natural compounds.With that concurrence, a lion's share (53%) of commercialized anticancer drugs are comprised of natural compounds since the 1980s [19].
In the present study, for the first time, we explored the inhibitory effects of a natural compound, DTN, on HCT-116 cells compared to 5-FU, while assessing its impact on the secretion of inflammatory cytokines.Our study showed that DTN inhibits HCT-116 cell viability at concentration and time-dependent manners.Similarly, previous studies also reported that DTN inhibits the viability of several cancer cells including prostate, liver, breast and colon with time and dose-dependent manners, which the magnitude of dead cells observed to be more eminent at higher dosage and time [11][12][13]20].Treatment of DTN on HT-29 cells yields the IC 50 value of 8.16 μg/ml [11], which was higher than presently on HCT-116 cells with 5.37 μg/ml at 24 h.Noteworthy, DTN exhibited a lower IC 50 value at 48 h of treatment compared to 5-FU indicating its potency to induce cell death of HCT-116 cells at a lower concentration.Moreover, observation under an inverted microscope depicted that DTN triggered morphological changes related to apoptosis which was further validated by way of flow cytometry.
In general, apoptotic machinery involves two prominent pathways: intrinsic (mitochondrial) and extrinsic (death receptor).The mitochondrial pathway is triggered by the intracellular stimulus that prompts mitochondrial outer membrane permeabilization, which presents with loss of MMP, eventually leading to the release of cytochrome c that activates caspases-9 and -3.Meanwhile, the death receptor signalling is activated via binding of ligands which subsequently switches on the protein caspase-8 through the association of death effector domain (DED) with Fas-associated protein via death domain (FADD) that directly upregulates caspases-3 [21].The present study found that DTN upregulates pro-apoptotic caspase-8 and -9 proteins in HCT-116 cells which validate its involvement in both apoptotic pathways.This was also parallelly observed in HT-29 cells as hitherto reported [14].Meanwhile, 5-FU induces apoptosis prominently via activating the mitochondrial pathway, which has previously been justified to be caspase 9-dependent [22].This may explain the significantly higher activity of caspase-9 in 5-FU compared to DTN-treated cells in this study.
Activation of caspase-9 is a salient part of ROSmediated apoptosis which involves mitochondrial insults.ROS is highly genotoxic, which is sacredly renowned as an important anticancer effect of natural compounds [23].The present study found that DTN triggers the excessive accumulation of iROS which simultaneously impeded MMP and induced DNA damage in HCT-116 cells.Likewise, past literature has also demonstrated that DTN triggers mitochondrial leakage via actuating release of cytochrome c and accumulation of ROS, as well as induction of DNA damage in HT-29 cells [14,24].Moreover, the present study also depicted that DTN demonstrates anti-migration and colonies formation capacities.The latter was justified via clonogenic assay, which essentially evaluates the ability of cancer cells to grow into colonies after being exposed to anticancer agents.Cancer cells that survived may form into colonies and divide unlimitedly, which is referred to as reproductive viability, this is closely associated with cancer stem-like cells (CSC) properties that are notoriously responsible for the formation of resistant cells and cancer relapse [25].Meanwhile, the former was validated via wound healing assay, in which DTN inhibited wound gap closure of HCT-116 cells indicating impairment of migrating mechanics.This may indicate that DTN potentially possesses anti-metastatic characteristics, which was also observed in 5-FU treated cells.
Generally, the key regulator of the cell cycle is comprised of two distinct stages, namely interphase and M phase.These two stages are separated by gap phases (G1, S and G2) and a non-proliferative state known as G0.Copious of NCs could trigger cell cycle block, usually, via G0/G1 (inhibition of proliferation), S (alteration of DNA synthesis) and G2/M (damaging the DNA) blockages which are strictly dependent on their mode of action [26].The present data illustrated that DTN triggers foremost phase G0/G1 blocks indicating it alters the proliferative potential of HCT-116 cells.This finding, however, is not consistent with previous literature which reported that DTN induces cell cycle arrest mainly at G2/M phases on HT-29 cells [11].This paradox may be contributed by the mutation signatures of both cell lines.As previously mentioned, HCT-116 cells have wildtype p53 gene function while HT-29 cells have lack thereof.Long-standing notion expounds that wild-type p53 cells were reported to be usually sensitive to G0/G1 arrest when exposed to an anticancer agent that targets p53 [27].Since upregulation of p53 was also seen in other cell lines treated with DTN [14], this may reason for the discrepancy of cell cycle blockade signature between both cell panels when treated with DTN.Additionally, the data also showed that 5-FU causes majorly S phase lag.The principle underlying 5-FU's mode of action is, it interferes with the thymidylate synthase, a crucial enzyme in DNA synthesis [28].
Several lines of evidence showed that the tumour reciprocally reacts with immune cells in the TME via inflammatory cytokines that greatly drive its initiation, growth, progression and metastasis capacities [9].While it was evident that these inflammatory cytokines positively aid the outgrowth of tumour cells, the same goes for their roles in anti-tumour immunity.Aligned with this notion, cytokines released during cell demise can ultimately trigger the activation of a robust immune response against tumours or dampen thereof [29].The present study explored the cytokines released during cell demise triggered by DTN and 5-FU on HCT-116 cells.Results illustrated elevation of several cytokines such as Th1 (IL-2, IL-6, TNF-α and IFN-γ ), immunosuppressive IL-10 and Th17 (IL-17A) against control.Compared to 5-FU, DTN triggers the higher secretion of IFN-γ and TNF-α, while IL-6 and IL-17A were secreted more in 5-FU treated cells.This demonstrates that DTN might exert the ability to induce immune response via ICD since IFN-γ was acknowledged as the most salient cytokine involved in the maturation of dendritic cells [30].IL-6 and TNF-α were validated to possess the aptitude to elevate expression of MHC class I protein which subsequently aided T-cells differentiation and activation of natural killer (NK) cells [29].Inversely, IL-6 was also reported to exhibit the capacity to enhance CRC cells invasion, which positively augments the occurrence of metastasis [31].Moreover, it drives 5-FU resistance and 5-FU mediated intestinal mucositis in CRC models [32].Thus, excessive secretion of the IL-6 may result in negative repercussions, which has been acknowledged as an important element of 5-FU-mediated inflammation.Besides, the role of IL-17A during cell demise is scarce; nevertheless, it was closely associated as a promoter of cancer progression in CRC [33].
In conclusion, we found that DTN triggers activation of both the intrinsic and extrinsic apoptotic pathways via accumulation of excessive iROS.This is simultaneously presented with cell cycle arrest, anti-migration and anti-colony formation capacities.Strikingly, unlike 5-FU, DTN induced potent secretion of Th1-related cytokines such as IFN-γ and TNF-α and lower secretion of pro-inflammatory IL-6 and Th17 which propose the possibility of it to evoke anti-tumour immune responses related to ICD while preventing excessive unwanted inflammation which provides the notion of DTN as a potential lead compound against CRC and warranted further validation in animal models.

Disclosure statement
No potential conflict of interest was reported by the author(s).
(A)), which was similarly demonstrated by 5-FU treated HCT-116 cells (Figure1(B)).The impact of DTN and 5-FU on HPC cells was not prominent, as more retainment of cell viability was observed even at the higher concentration (Figure1(C,D), respectively).At 24 h, DTN exhibited an IC 50 , a value of 5.37 μg/ml, which is not significantly different compared to 5-FU (5.61 μg/ml).While at 48 h, notably, DTN exerted a lower IC 50 value of 1.90 μg/ml compared to 5-FU (2.61 μg/ml, p < .05).Treatment of HPC cells with DTN exhibited IC 50 values of 38.1, 18.0 μg/ml, while 5-FU demonstrated IC 50 values of 23.2 and 10.4 μg/ml at the respective time points of 24 and 48 h.This decisively showed that DTN is less toxic on HPC cells compared to 5-FU (Figure1(E)).

Figure 1 .
Figure 1.Cytotoxicity effect of DTN and 5-FU (0-20 μg/ml) on HCT-116 and HPC cells.(A) Effect of DTN on cell viability of HCT-116 cells.(B) Effect of 5-FU on cell viability of HCT-116 cells.(C) Effect of DTN on cell viability of HPC cells.(D) Effect of 5-FU on cell viability of HPC cells.(E) The IC 50 concentration of DTN and 5-FU treated HCT-116 and HPC cells at 24 and 48 h.Data shown are mean of three independent experiments.* P < .05indicates significant difference.DTN: Dentatin; 5-FU: 5-fluorouracil.

Figure 3 .
Figure 3. Impact of DTN (1.90 μg/ml) and to 5-FU (2.61 μg/ml) on cell migration and colonies formation of HCT-116 cells via wound healing and clonogenic assays, respectively.(A) Photographs of treatment of DTN and 5-FU at their respective IC 50 concentration at 48 h on wound healing assay at 0, 24, 48 and 72 h under inverted microscope.(B) Graph of wound-gap closure per time point of DTN and 5-FU treated HCT-116 cells.(C) Photographs under inverted microscope of DTN and 5-FU treated cells for 48 h at their respective IC 50 concentration.(D) Graph of colonies count of DTN and 5-FU treated HCT-116 cells.Data shown are mean of three independent experiments.* P < .05indicates significant difference.DTN: Dentatin; 5-FU: 5-fluorouracil.