Clausenidin upregulated p53 and caused apoptosis in HT-29 tumor cell lines

1 MAKNA Cancer Research Laboratory, Institute of Bioscience, University Putra Malaysia, Serdang, Selangor, Malaysia 2 Department of Biochemistry, Kaduna State University, Main Campus, PMB 2336, Kaduna, Nigeria. 3 Department of Veterinary Pathology and Microbiology, Faculty of Veterinary, University Putra Malaysia, Serdang, Selangor, Malaysia. 4 Laboratory of Vaccine and Therapeutics, Institute of Bioscience, University Putra Malaysia, Serdang, Selangor, Malaysia. 5 Department of Chemistry, Faculty of Science, University Putra Malaysia, Serdang, Selangor, Malaysia.


INTRODUCTION
The protein, P53 (TP53) was first identified in 1979 and named after its molecular weight, which is 53 kDa (Levine et al., 1991).The protein, P53, is one of the best-known tumor suppressor protein that is encoded by the TP53 gene located on the short-arm of chromosome 17.Besides its involvement in the induction of apoptosis, it is also a key regulator of DNA recombination, gene amplification, segregation of chromosomes, cell cycle and differentiation and cellular senescence (Oren and Rotter, 1999).Deletion of the p53 gene in cells leads to the decrease in rate of apoptosis (Slatter et al., 2011;Vikhanskaya et al., 2007).Because this protein play a profound role in the maintenance of cell integrity, it is often referred to as 'guardian of the genome' (Lane, 1992).In human cancers, the p53 gene is generally impaired (Bai and Zhu, 2006).P53 can detect DNA damage and direct the cells to undergo DNA repair or apoptosis.Clausena excavata Burm f. is a native of the Asian tropical forest commonly found in India, Thailand and Malaysia (Huang et al., 1997;Manosroi et al., 2004).The plant is claimed to have numerous therapeutic benefits that includes anti-bacteria, anti-fungal, anti-inflammatory (Wu et al., 1994) and anticancer effects (Manosroi et al., 2004;Arbab et al., 2013) but its detailed mechanism of action is yet to be understood.Thus far, coumarins and alkaloids are the major bioactive components to have been isolated from C. excavata.Previous study reported that clausenidin (Figure 1) isolated from C. excavata induces caspase-dependent cell death in colon cancer cell line (Waziri et al., 2016).Since p53 is the most frequent mutated gene in cancerous cells, the current study investigates its role in clausenidin-treated HT-29 cells.Specifically, the study aims to unravel the effect of clausenidin on p53-dependent-cell cycle arrest andapoptosis in colon cancer cell line.

Cell line and cell culture
HT-29 cell line was purchased from American Type Culture Collection (ATCC) and maintained in DMEM medium supplemented with 10% fetal bovine serum (FBS).The cells were grown in an incubator at 37°C and 5% CO2.

Scanning electron microscopy (SEM)
To prepare the cells for SEM, HT-29 cells were seeded at a density of 1 × 10 6 cells/T-25 mL flask overnight, and treated with the IC50 of clausenidin (13.8 µg/mL) for 24, 48 and 72 h while the negative control cells were treated with 0.1% (v/v) DMSO.At the end of the treatment period, cells were harvested with trypsin and washed three times with PBS by centrifugation at 1000 x g for 10 min at 4°C before fixing with 4% glutaraldehyde and 1% osmium tetraoxide for 6 and 2 h respectively at 4°C.The cells were washed three times (10 mins each) after each fixing with 0.1 M sodium cacodylate buffer and centrifuged at 1000 x g for 5 min to collect the cell pellets followed by successive dehydration with 35, 50, 75 and 95% acetone (10 min each).The cells were further dehydrated three times (15 min each) with 100% acetone before drying for 30 min on a critical drier.Finally, the cells were placed on stubs, coated with gold particles and then viewed under the JSM 6400 scanning electron microscope (Joel, USA).

Transmission electron microscopy (TEM)
For TEM, cells (1 × 10 6 cells/flask) were seeded overnight in T-25 mL flask and treated with the IC50 of clausenidin (13.8 µg/mL) for 24, 48 and 72 h while negative control cells were treated with 0.1% (v/v) DMSO.The cells were processed by fixing with 4% glutaraldehyde and osmium tetraoxide, and dehydrated with acetone as described previously.Thereafter, infiltration was done with acetone:resin mixture successively for 1 and then 3 h in a ratio of 1:1 and 1:3, respectively.Further infiltration was done overnight with 100% resin, and the cells were embedded by inserting into a beam capsule containing the resin.The embedded samples were polymerized in an oven at 60°C for 2 days before cutting into thick sections (1 µm) using an ultramicrotome.The thick sections were stained with toluidine blue and cut further into thinner sections of 60 to 90 nm that were stained further with uranyl acetate and lead for 15 and 10 min, respectively.The fully stained sections were then viewed under the H-7100 transmission electron microscope (Hitachi, Japan).

Cell cycle assay
The cell cycle was monitored by flow cytometry (Becton Dickinson, USA) using the CycleTest TM plus DNA reagent kit according to manufacturer's protocol.The cells were seeded overnight at a density of 1 × 10 6 cells/flask in a T-25 mL flask and treated with 5, 15, 30 and 40 µg/mL of clausenidin while negative control cells were treated with 0.1% (v/v) DMSO, both for 24 h.The cells were then washed twice with PBS and centrifuged at 1000 × g for 5 min at 4°C and the supernatant discarded after each washing.The pellets were suspended in 250 µL of solution A (trypsin buffer), mixed gently and incubated at room temperature for 10 min.Then 200 µL of solution B (trypsin inhibitor and RNase) was added and the cells incubated for another 10 min at room temperature.Finally, 200 µL of ice-cold solution C (125 µg/mL PI final conc.) was added to each reaction tube and the cells incubated on ice for 10 min in the dark and analysed by flow cytometry (BD FACS, Calibur).

Annexin-V assay
Annexin-V assay was performed using the FITC Annexin-V Apoptosis Detection Kit I (BD Pharmingen, US) according to manufacturer's instruction.Briefly, cells were seeded in a 6-well plate at a density of 3 × 10 5 cells/well and treated separately with 5, 15, 30 and 40 µg/mL of clausenidin while negative control cells were treated 0.1% (v/v) DMSO both for 24 h.After harvesting with trypsin, cells were washed twice with PBS and suspended in 5 µL each of annexin-V and PI, vortexed gently and incubated in the dark for about 15 min.This was followed by the addition of 400 µL of binding buffer to each reaction tube and the cells were analyzed by flow cytometry (BD FACS, Calibur).

Gene expression studies
The extraction of RNA was basically done to determine gene expressions of clausenidin-treated cells.

RNA isolation
The cells were seeded overnight in T-25 mL flask at a density of 1 × 10 6 cells/well and treated with 13.8 µg/mL clausenidin for 12 or 24 h.Negative control cells were treated with 0.1% (v/v) DMSO for 24 h.The cells were then harvested after detachment with trypsin and washed with PBS.The RNA was extracted using Total RNA extraction kit (GF-1 TRE kit, Vivantis technologies) according to the manufacturer's protocol and its purity determined and amount quantified at 260nm in the nanodrop spectrophotometer (Eppendorf, United Kingdom).

Primer design
The primers for the genes of interest and housekeeping gene used in this study were designed on the NCBI website using PRIMER-BLAST software (Table 1).The primers were purchased from Biosune (Shanghai, China), while the internal control (Kanr) was supplied by Beckman Coulter (USA).The quality of the designed primers was checked using the Oligocal software.

RT-qPCR
The reverse transcriptase quantitative PCR (RT-qPCR) was carried out according to the GenomeLab GeXP Kit (Beckman Coulter, USA) protocol, in an XP Thermal Cycler (Bioer Technology, Germany).Reverse transcription (RT) and PCR were done according to manufacturer's instructions.The conditions were set as follows; RT reaction was at 48°C for 1 min; 37°C for 5 min; 42°C for 60 min; 95°C for 5 min; then held at 4°C, while PCR was as follows: Initial denaturation at 95°C for 10 min, followed by two-step cycles of 94°C for 30 s and 55°C for 30 s, ending in a single extension cycle of 68°C for 1 min.The PCR products were finally analyzed on the GeXP genetic analysis system and the results normalized on express Profiler software.The β-actin gene was used for normalization.

Protein expression studies
To further ascertain the findings of the gene expression analysis, protein profile array and Western blot assays were done to determine expression of apoptotic protein in clausenidin-treated HT-29 cells.

Human apoptosis protein profile array
The protein profile array was performed using the proteome profiler antibody array kit (Raybiotech Inc., USA) according to the manufacturer's protocol.Briefly, the cells were seeded overnight at a density of 1 × 10 6 cells/well and treated with 15 µg/mL clausenidin while negative control cells were treated with 0.1% (v/v) DMSO, both for 24 h.Thereafter, protein samples from clausenidin-treated HT-29 cells were incubated overnight at 4°C on primary antibody coated slides (Raybiotech Inc., USA) and washed with wash buffer I before incubating with the biotin-conjugated antibody at 25°C for 2 h.The slides were washed with wash buffer I, incubated with 1,500fold diluted Hilyte Plus TM -conjugated streptavidin in the dark for 2 h and washed again with wash buffer I before disassembling the slides from the incubation frame and chamber.Further washing of slides was done with wash buffer II for 30 min and air-dried for about 1 h and then the result analyzed using the analysis tool software (RayBio Human Apoptosis Array C-series 1).

Protein assay
The cells were seeded in a 6-well plate overnight at a density of 5 × 10 5 cells/well before treating with either 5, 15, 30 or 40 µg/mL clausenidin while negative control cells were treated with 0.1% (v/v) DMSO, both for 24 h.The cells were washed with PBS by centrifugation at 5000 × g for 5 min at 4°C.The cell pellets were suspended in 200 µL RIPA buffer (Thermo Fisher scientific, USA) for 30 min, on ice with vortexing at 10-minute intervals for 20 s.The cell suspension was then centrifuged at 14000 × g for 25 min at 4°C.The supernatant collected were transferred to fresh tubes.To quantify protein, 250 µL Bradford reagent was added to 5 µL sample in a 96-well plate and the plate incubated for 5 min at room temperature in the dark.A protein standard curve was prepared with bovine serum albumin in PBS.The absorbance was determined at 570 nm in an ELISA plate reader (BioTek, USA) and protein concentration of samples obtained from the standard curve.

SDS-PAGE and Western blot
Protein samples were separated by electrophoresis for 90 min at 120 V on a 10% sodium doedecyl sulphate-polyacrylamide gel.The samples were run using Tris running buffer (25 mM Tris base, 192 mM glycine, 0.1% SDS, pH 8.3) medium.The resolved proteins from SDS-PAGE gel were transferred to the polyvinylidene difluoride (PVDF) membrane (Biorad, USA) using the wet transfer method (Gels, 2001).The transfer of proteins was done with the use of transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) at 100V for 90 min.The membrane was then blocked for 2 h with 5% skim milk in an orbital shaker (Heidolph, Germany) before washing trice for 10 min each time with PBS-Tween 20 on an orbital shaker (Heidolph, Germany).Primary and secondary antibodies (Santa Cruz, USA) were prepared in 5% skim milk at a concentration of 1:2000 and 1:3000 respectively.The membranes were incubated with the primary antibodies at 4°C for 24 h followed by secondary antibody at 25°C for 1 h.The membranes were washed trice for 10 min each time with PBS-Tween 20 after incubation with either primary or secondary antibodies.The membranes were developed using the chemiluminescent substrate (ThermoFisher Scientific, USA) according to manufacturer's protocol.Solutions A and B were mixed in equal proportions and 1 mL of the mixture was added to the membrane while ensuring the surface is completely covered with the substrate.The membranes were incubated for 5 min and then viewed on a ChemiDoc TM imaging system (Biorad, USA) and the protein expressions determined.

Statistical analysis
One way analysis of variance (ANOVA) was used to determine the level significance at 95% confidence interval (p<0.05) using the Waziri et al. 1415 SPSS 22 software (SPSS Inc, Chicago IL, USA).

Effect of clausenidin on cell morphology and nuclear fragmentation
Morphological aberrations of clausenidin-treated cells were examined using scanning electron (SEM) and transmission electron microscopy (TEM).The SEM analysis reveals features of apoptosis that includes membrane blebbing and cytosolic modifications (Figure 2).Further investigation using TEM showed chromatin condensation and margination, convolution of nuclear outline as well as vacuolation (Figure 3).The fragmentation of nucleus was observed at 48 and 72 h of treatment with clausenidin (Figure 3C-D).

Clausenidin inhibited the proliferation of HT-29 cells and caused a G0/G1 cell cycle arrest
The effect of clausenidin on cell cycle progression in HT-29 cells was investigated by flow cytometry.It was observed that clausenidin inhibited the proliferation of HT-29 cells and also caused a G0/G1 arrest (Figure 4F).The cell cycle distribution shows an increase in the percentage of sub G0/G1 cells (apoptotic cells) after treatment with clausenidin in a dose dependent manner.Furthermore, clausenidin caused a significant decrease (p<0.05) in the proportion of viable cells.

Clausenidin induced apoptosis in HT-29 cells
Clausenidin treatment caused a significant (p<0.05)dose dependent apoptosis in HT-29 cells after 24 h of treatment (Figure 5).In addition, the viable cells decreased significantly (p<0.05) in a dose dependent manner after treatment with clausenidin for 24 h.

Gene expression studies
The gene expression studies showed the effect of clausenidin on mRNAs that regulate cell cycle in HT-29 cells.Clausenidin caused over 10 fold increase in the expression of p53 mRNA after 12 hours of treatment.
Similarly, the treatment significantly (p<0.05)upregulated the expressions of cyclins A, D and E mRNAs at 12 and 24 h of treatment (Figure 6).

Protein expression studies
The analysis of the apoptotic pathway related proteins showed that clausenidin upregulated the expression of   the pro-apoptotic proteins and down-regulated the expression of the anti-apoptotic proteins (Table 2).The only exceptions are the anti-apoptotic proteins, HSP 60 and 70 that were upregulated following clausenidin treatment.More importantly, the upregulation of p53 and p27 were observed and this confirms the finding of out gene expression analysis.In addition, the Western blot analysis was performed to further validate the findings of the protein profile array.It was observed that clausenidin significantly increased and decreased the expression of the pro-apoptotic bax, and JNK, and anti-apoptotic, bcl 2 proteins respectively (Figure 7).

DISCUSSION
The traditional use of Clausena excavata and its compounds for the treatment of cancer is based on Asian folklore that is devoid of scientific evidence.Previous study reported the anti-tumor properties of clausenidin isolated from the root of C. excavata (Waziri et al., 2016).However, the detailed mechanism of anti-cancer cell effect of clausenidin is poorly understood.The current study investigates apoptosis mediated by p53 in clausenidin-treated HT-29 cells.
Apoptosis is a coordinated cell death process that is accompanied by changes in the morphology of tumor cells that affects both cytoplasm and nucleus.This process usually takes several hours depending on the cell type and initiating apoptotic stimuli (Häcker, 2000), and begins with chromatin condensation and fragmentation of the nucleus as well as cellular shrinkage (Kroemer et al., 2005).Furthermore, the chromatin moves to the margin or periphery of the nucleus and condenses further until it breaks up inside into smaller materials (Majno and Joris, 1995).Our TEM micrographs material to the margins of the reveals the migration of the condensed chromatin nucleus.Also, the fragmentation of the nucleus progressed with time reaching its peak at 72 h.In addition, the SEM micrograph showed membrane blebs and cytosolic destruction of the clausenidin-treated HT-29 cells.Membrane blebbing, cytosolic damage and loss of membrane integrity are events that characterize the latter stages of apoptosis (Kroemer al., 2005).
P53 is a tumor suppressor that promotes apoptosis via transcription-dependent or -independent mechanisms in cells (Fridman and Lowe, 2003).The loss of the p53 gene function contributes to carcinogenesis and tumor cell survival (Béroud and Soussi, 2003;Hussain and Harris, 1998) (Hartwell and Kastan, 1994).In this study, clausenidin affected the progression of cell cycle by causing a G0/G1 cell cycle arrest in the treated HT-29 cells.The induction of cell cycle arrest is an indicator of apoptosis (Kummalue et al., 2007).On the other hand, the activation of p21 gene by p53 comprises one of the main surveillance mechanisms in cell cycle regulation (El-Deiry et al., 1994).For the cell cycle to progress from G1 to S phase, there must be accumulations of cyclins A, D and E that activate the cyclin dependent kinases (CDKs) (Sherr, 1996).The p53 gene transactivates target genes like p21, which is an inhibitor of cell cycle.Increased expressions in the p53 and p21 mRNAs were observed in the clausenidintreated HT-29 cells.At the same time, there were also increased expressions of the cyclins A, D and E in the treated cells.The upregulation of the cyclins in the clausenidin-treated HT-29 cells was countered by the significant upregulation of p53 and p21 genes that prevented the progression of the cell cycle from G1 to S phase.However, based on the findings, it was postulated that one of the mechanism of action of clausenidin on the HT-29 cells is through the activation of p53 that increased expression of p21 leading to G0/G1 phase cell cycle arrest.Similarly, p53 transactivates the pro-apoptotic members of the bcl 2 family, bax (Miyashita et al., 1994) and bid (Sax et al., 2002).In this study, significant expressions of bax and bid in clausenidin-treated HT-29 cells that increased the ratio of pro-to anti-apoptotic bcl 2 proteins and subsequent activation of apoptosis was observed.The ultra-structural micrographs observed in this study confirm the execution of apoptosis in the clausenidin-treated HT-29 cells.More so, p53 is known to prevent the expression of cell survival factors and inhibitors of apoptosis (IAPs).The anti-apoptotic protein, survivin is another target of p53 that encodes an IAP, which promotes cell survival (Ambrosini et al., 1997).
Clausenidin treatment caused a decreased expression of survivin in HT-29 cells which we presumed to be by the significant expressions of p53.Furthermore, decreased expressions of the anti-apoptotic proteins, bcl 2, bcl w and HSP 27 were observed which we suspected may have been triggered by p53 in the clausenidin treated HT-29 cells.The non-transcriptional roles of p53 involves mitochondrial activity, eviction of cytochrome c and the activation of caspases among others (Mihara et al., 2003).This was observed in our previous study on clausenidin-treated HT-29 cells.
In conclusion, the current study suggests that bax, survivin and p21 are the main effectors of p53 mediated

Figure 7 .
Figure 7. Protein expression in HT-29 cells after treatment with various concentrations of clausenidin.(I) Western blot; (II) Relative protein expression.*Means significantly (p<0.05)different from control.

Table 1 .
Gene and primer sequences used in the assay.