Hydroxychavicol, a polyphenol from Piper betle leaf extract, induces cell cycle arrest and apoptosis in TP53-resistant HT-29 colon cancer cells

Hydroxychavicol, a polyphenol from Piper betle leaf extract, induces cell cycle arrest and apoptosis in TP53 -resistant HT-29 colon cancer cells 从花椒叶中提取的羟基胡椒酚可以诱导耐TP53的HT-29结肠癌细胞的细胞周期阻滞和凋亡 1 first-author 2 2 1 3 corresp Mee Lee LOOI, meelee.looi@um.edu.my Mee Lee LOOI, meelee.looi@um.edu.my meelee.looi@um.edu.my https://orcid.org/0000-0001-5415-6973 School of Biosciences, Taylor’s University, Lakeside Campus, 47500 Subang Jaya, Malaysia School of Biosciences, Taylor’s University, Lakeside Campus, 47500 Subang Jaya, Malaysia Forest Research Institute Malaysia, 52109 Kepong, Malaysia Forest Research Institute Malaysia, 52109 Kepong, Malaysia Department of Biomedical Science, Faculty of Medicine, University of Malaya, 50603Kuala Lumpur, Malaysia Department of Biomedical Science, Faculty of Medicine, University of Malaya, 50603Kuala Lumpur, Malaysia This study aims to elucidate the antiproliferative mechanism of hydroxychavicol (HC). Its effects on cell cycle, apoptosis, and the expression of c-Jun N-terminal kinase (JNK) and P38 mitogen-activated protein kinase (MAPK) in HT-29 colon cancer cells were investigated. HC was isolated from Piper betle leaf (PBL) and verified by high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and gas chromatography-mass spectrometry (GC-MS). The cytotoxic effects of the standard drug 5-fluorouracil (5-FU), PBL water extract, and HC on HT-29 cells were measured after 24, 48, and 72 h of treatment. Cell cycle and apoptosis modulation by 5-FU and HC treatments were investigated up to 30 h. Changes in phosphorylated JNK (pJNK) and P38 (pP38) MAPK expression were observed up to 18 h. The half maximal inhibitory concentration (IC 50 ) values of HC (30 μg/mL) and PBL water extract (380 μg/mL) were achieved at 24 h, whereas the IC 50 of 5-FU (50 μmol/L) was obtained at 72 h. Cell cycle arrest at the G 0 /G 1 phase in HC-treated cells was observed from 12 h onwards. Higher apoptotic cell death in HC-treated cells compared to 5-FU-treated cells ( P < 0.05) was observed. High expression of pJNK and pP38 MAPK was observed at 12 h in HC-treated cells, but not in 5-FU-treated HT-29 cells ( P < 0.05). It is concluded that HC induces cell cycle arrest and apoptosis of HT-29 cells, with these actions possibly mediated by JNK and P38 MAPK. 目的 2 阐明羟基胡椒酚（HC）的抗恶性细胞增生的机制。并研究HC对HT-29结肠癌细胞的细胞周期、凋亡及c-Jun氨基末端激酶（JNK）和P38丝裂原活化蛋白激酶（MAPK）表达的影响。 方法 2 从蒌叶（PBL）中分离出HC，经高效液相色谱（HPLC）、核磁共振（NMR）和气相色谱-质谱（GC-MS）进行检测。在处理24、48和72 h后，检测标准药物5-氟尿嘧啶（5-FU）、PBL水提物和HC对HT-29细胞的细胞毒性作用。检测30 h内5-FU和HC处理对细胞周期和凋亡的调控作用。同时检测18 h内磷酸化JNK（pJNK）和磷酸化P38（pP38）MAPK的表达变化。 结果 2 HC（30μg/ mL）和PBL水提物（380μg/ mL）的半数最大抑制浓度（IC 50 ）值在24 h时达到，而5-FU（50μmol/ L）的IC 50 值在72 h时达到。从12h开始HC处理的细胞停滞在细胞周期的G 0 /G 1 期。与5-FU处理的细胞相比，HC处理的细胞凋亡率更高（ P < 0.05）。在HC处理的细胞中，pJNK和pP38 MAPK在12 h时出现高表达，而在5-FU处理的HT-29细胞中则没有（ P < 0.05）。 结论 2 由此可见，HC可诱导HT-29细胞的细胞周期阻滞和凋亡，这些作用可能由JNK和P38 MAPK介导。 Piper betle Hydroxychavicol (HC) Cell cycle Apoptosis c-Jun N-terminal kinase (JNK) P38 mitogen-activated protein kinase (MAPK) 蒌叶 羟基胡椒酚（HC） 细胞周期;细胞凋亡 c-Jun氨基末端激酶（JNK） P38丝裂原活化蛋白激酶（MAPK） 1 Introduction 1 Plants and natural products have been investigated extensively for centuries, focusing on drug discovery and development. Anticancer agents such as vincristine, vinblastine, and paclitaxel are examples of drugs derived from plants and natural products ( Amin et al., 2009 Amin et al., 2009 ; Bhanot et al., 2011 Bhanot et al., 2011 ; Batra and Sharma, 2013 Batra and Sharma, 2013 ). Piper betle leaves (PBLs) have been used for centuries throughout Asia, and by ayurvedic practitioners for the prevention and treatment of cancers ( Garodia et al., 2007 Garodia et al., 2007 ). While betel quid (BQ) chewing was found to be carcinogenic and BQ components were shown to induce inflammatory response in oral mucosal cells ( Jeng et al., 2000 Jeng et al., 2000 , 2001 2001 ; Trivedy et al., 2002 Trivedy et al., 2002 ). PBL was shown to have antioxidant, antimutagenic, anticarcinogenic, antiplatelet, and anti-inflammatory effects ( Jeng et al., 2002 Jeng et al., 2002 ; Chang et al., 2007 Chang et al., 2007 ; Kumar et al., 2010 Kumar et al., 2010 ; Gundala and Aneja, 2014 Gundala and Aneja, 2014 ). In the past, various studies revealed the potential cytotoxic effect of PBL extracts on various cancer cells such as colon ( Ng et al., 2014 Ng et al., 2014 ), cervical ( Widowati et al., 2013 Widowati et al., 2013 ), breast ( Abrahim et al., 2012 Abrahim et al., 2012 ), and prostate ( Abrahim et al., 2012 Abrahim et al., 2012 ). Among the many bioactive compounds of PBL, hydroxychavicol (HC) has been the most widely reported for its cytotoxic effect ( Kumar et al., 2010 Kumar et al., 2010 ; Gundala et al., 2014 Gundala et al., 2014 ). Studies have shown its involvement in reactive oxygen species (ROS) generation ( Gundala et al., 2014 Gundala et al., 2014 ), DNA damage ( Chen et al., 2000 Chen et al., 2000 ), cell cycle deregulation and apoptosis ( Chang et al., 2002 Chang et al., 2002 ; Jeng et al., 2004 Jeng et al., 2004 ; Chakraborty et al., 2012 Chakraborty et al., 2012 ; Rahman et al., 2014 Rahman et al., 2014 ). Abnormalities in cell proliferation and the evasion of programmed cell death (apoptosis) are the two prominent hallmarks of cancer ( Hanahan and Weinberg, 2011 Hanahan and Weinberg, 2011 ). Disruptions in cell signaling pathways, such as the mitogen-activated protein kinase (MAPK) pathway, play an important role in cancer development and progression ( Dhillon et al., 2007 Dhillon et al., 2007 ). Among the MAPK family proteins, the c-Jun N-terminal kinase (JNK) and P38 MAPK are associated with colon cancer, whereas the extracellular signal-regulated kinase (ERK) is linked with rectal cancer ( Slattery et al., 2012 Slattery et al., 2012 ). Various fundamental cellular processes involved in cancer progression, such as apoptosis, proliferation, differentiation, motility, stress response, and survival, are controlled by the JNK and P38 MAPK signaling pathways ( Wagner and Nebreda, 2009 Wagner and Nebreda, 2009 ). Many reports associated the disruption of cell cycle and induction of apoptosis in cancer cells by natural products with the signal transduction regulation ( Sarkar et al., 2009 Sarkar et al., 2009 ; Chakraborty et al., 2012 Chakraborty et al., 2012 ; Angulo et al., 2017 Angulo et al., 2017 ). The first-choice chemotherapy drug for colon cancer has been 5-fluorouracil (5-FU); however, it has limited effectiveness due to its short biological half-life ( Wigmore et al., 2010 Wigmore et al., 2010 ). It exerts its effect via tumor protein p53 ( TP53 ) activation ( Longley et al., 2003 Longley et al., 2003 ), though only approximately 40%‒50% of sporadic colorectal cancer cases are associated with TP53 mutation ( Takayama et al., 2006 Takayama et al., 2006 ). Drug resistance has been a great challenge in the treatment of colon cancer. Any compounds that can exert an inhibitory effect on TP53 mutant cells could therefore serve as potential drugs for this purpose. The HT-29 cell line is a model of TP53 -mutated cells, hence it is an excellent candidate for studying the effects of potential chemotherapeutic agents on colon cancer. This study was designed to explore the antiproliferative potential of PBL water extract and its active compound HC on HT-29 cells. The modulation effects of HC on the cancer cell cycle, apoptosis, as well as JNK and P38 MAPK protein expression, were further investigated. 2 Materials and methods 1 2.1 Sample collection 2 A total of 10 kg of PBL was collected in Simpang Morib, Selangor, Malaysia. Samples of leaves and fruits were sent to the Plant and Herbs Herbarium at the Forest Research Institute of Malaysia (FRIM) for verification. Leaves were subjected to further processing. They were washed and dried in oven (Memmert, Schwabach, Germany) overnight at 60 °‍C until the moisture level was below 10%. The dried PBLs were ground, sealed, and stored in an airtight container at 25 °C until further use. 2.2 Water extraction of PBL 2 Water extraction from PBL was performed with a 30-L pilot extractor for herbs (Technoniaga Sdn Bhd, Kuala Lumpur, Malaysia). Extraction was carried out and monitored at 60 °‍C. The ratio of dried PBL to water was 1:20 (mass:volume, kg/L) with extraction running for 1 h. Thereafter, the water extract was filtered and filtrates were freeze-dried in a freeze drier (Virtist Genesis, New York, USA). Freeze-dried samples (porous powder) were stored at -20 °C. 2.3 HC isolation 2 The freeze-dried crude PBL water extract (10.0 mg) was dissolved in 20 mL of water and 30 mL of methanol (analytically pure (AR) grade), and then filtered on 140 g of prepacked MCI-Gel ® column (Mitsubishi Chemical Corporation, Tokyo, Japan) conditioned with 50% water. The eluent used was 100 mL of 50%‒100% methanol in distilled water at various concentrations. Each fraction of about 50 mL was collected and simultaneously spotted on a thin layer chromatography (TLC) plate along with the HC standard. The solvent system used for TLC was hexane (AR grade) and ethyl acetate (AR grade) in the volume ratio of 7:3. The fractions containing HC were pooled and dried using a rotary evaporator (Hahn Shin Science, Gyeonggi-do, Korea). Thereafter, column chromatography, with silica gel as the stationary phase, was performed to further isolate HC. The dried sample was loaded evenly onto the column containing silica gel, and a fraction collector (Advantec, Southampton, UK) was attached to the column. The volume ratio of eluent used was 8:2 of hexane (AR grade) and ethyl acetate (AR grade). As the fractions were collected, TLC was performed for each one. Fractions with HC were pooled and further dried using a rotary evaporator. Part of the isolated HC was sent for validation, while the remaining amount was stored at -80 ℃. 2.4 HPLC 2 Isolated HC was injected into high-performance liquid chromatography (HPLC) to determine the chemical profile and to quantify concentration. Standards of HC were prepared with methanol in concentrations ranging from 20 to 2 000 ppm (part per million). The injection volume was 10 µL. The HPLC analyses were carried out using a Waters 600E System Controller (Waters Corporation, Massachusetts, USA) coupled with a Waters 996 Photo iodide Array Detector (Waters Corporation). The stationary phase was Phenomenex Luna C18 100 A (250 mm×4.6 mm, 5 μm particle size). Elution was performed using a gradient system consisting of a mixture of 0.1% formic acid in Milli-Q water and 100% acetonitrile. The flow rate of the mobile phase was set at 1 mL/min at room temperature. Chromatograms were acquired at 280 nm for 20 min. 2.5 NMR 2 The nuclear magnetic resonance (NMR) spectra were obtained from a JEOL ECX500 FT NMR Spectrometer system (JEOL Ltd., Tokyo, Japan). Deuterated chloroform (CDCl 3 ) was used to dissolve HC. The chemical shifts from the spectra were recorded in terms of ppm, and coupling constants ( J ) were given in Hertz (Hz). 2.6 GC-MS 2 Mass spectra were recorded with electron ionization mass spectrometry (EIMS) using a direct injection probe on a Shimadzu gas chromatography-mass spectrometry (GC-MS) QP 5050A Spectrometer (Shimadzu Corporation, Kyoto, Japan). 2.7 Cell line and cell culture maintenance 2 The HT-29 cells were purchased from American Tissue Culture Collection (ATCC, Manassas, USA). They were cultured in 5% carbon dioxide at 37 °C in McCoy’s 5A medium modified with L-glutamine supplemented with 10% fetal bovine serum (FBS). During passages, cells were washed with phosphate-buffered saline (PBS) and trypsinised with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) for 3 min. Following the detachment of cells, culture medium was added to neutralize the trypsin-EDTA. Cells were then centrifuged (4 °‍C, 1 000 r/min, 5 min), the supernatant was discarded and the pellet was resuspended in culture medium. Cell enumeration was performed in a hemocytometer via the trypan blue exclusion method. An appropriate number of cells were then seeded for either propagation or treatment. 2.8 MTS assay 2 There were three treatment groups: (1) 5-FU (25‒200 µmol/L), (2) crude PBL water extract (50‒800 μg/mL), and (3) HC (10‒50 µg/mL). Each treatment solution was freshly prepared on the day of treatment; treatment effect was observed up to 72 h. A total of 1×10 4 cells/well were seeded in a 96-well plate. The plate was incubated at 37 °‍C for 24 h. Thereafter, cells were treated with respective treatments. The duration of treatment was set at 24, 48, and 72 h for (1) 5-FU, and 24 and 48 h for (2) crude PBL water extract and (3) HC. At the end of each respective treatment, 20 μL of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) solution was added to each well, wells were gently shaken and incubated at 37 °‍C for 1 h. Subsequently, the MTS product was measured at 490 nm using a microplate reader (BioTek, Winooski, USA). Cell viability was calculated based on the equation below: cell viability=(absorbance of treated cells-absorbance of treatment alone)/(absorbance of untreated cells-absorbance of media alone)×100%. 2.9 Cell cycle analysis 2 The seeding of HT-29 cells was performed in a 6-well plate at a concentration of 3×10 5 cells/well for 24 h. To observe the cell cycle event, cells were treated with the respective half maximal inhibitory concentration (IC 50 ) values of HC (30 μg/mL) and 5-FU (50 μmol/L) for 12, 18, 24, and 30 h. Non-treated (NT) controls and treated cells were then harvested. Cells were centrifuged, the supernatant was discarded, and the cell pellet was washed with cold PBS twice and fixed with ice-cold 70% ethanol solution. The fixed cells were centrifuged, washed twice with cold PBS, and resuspended in 0.5 mL of 10 μg/mL RNase solution, followed by incubation for 30 min in the dark at 4 °‍C. Thereafter, 0.5 mL of 20 µg/mL propidium iodide (PI) solution was added and incubated in the dark at 4 °C for 30 min. Samples were then vortexed gently for the measurement of fluorescence excitation of PI (FL-2 signal) using the Accuri C6 flow cytometer (BD Biosciences, New Jersey, USA). The percentages of cell population in the different phases (G 0 /G 1 , S, and G 2 /M) of cell cycle were determined using the ModFit LT Version 4 software (Verity Software House, Topsham, USA). 2.10 Apoptosis assay 2 HT-29 cells were seeded in a 6-well plate at a concentration of 3×10 5 cells/well for 24 h. Cells were then treated with HC (30 μg/mL) and 5-FU (50 μmol/L) for 12, 18, 24, and 30 h. Samples were prepared as per manufacturer’s instructions (BD Biosciences). Briefly, control and treated cells were trypsinized, and cell pellets were resuspended with 1× Annexin-binding buffer (BD Biosciences) to a cell concentration below 1×10 6 cells/mL. Five microliters of Annexin V-fluorescein isothiocyanate (V-FITC) (1× solution provided in the kit; BD Biosciences) and 5 μL of 20 μg/mL PI (BD Biosciences) were then added to a tube containing a total of 1×10 5 cells. Following the incubation of tubes at room temperature for 15 min, 400 μL of Annexin-binding buffer was added to each sample tube. Thereafter, cells were analyzed within an hour by flow cytometer using the FL-1 signal (V-FITC signal) and FL-2 signal (PI) in the Accuri C6 flow cytometer (BD Biosciences). A minimum of 1×10 4 events was collected for each sample, and the population of cells was characterized into different quadrants (dot plot) using the CFlow Plus analysis software (BD Biosciences). 2.11 Protein extraction and quantification 2 A total of 3×10 5 cells per well were seeded in a 6-well plate for 24 h, followed by treatments with HC (30 μg/mL) and 5-FU (50 μmol/L). The cells were harvested at 12 and 18 h. The cell pellet was washed twice with cold PBS. Subsequently, 60 μL of radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor (5%) and phosphatase inhibitor (5%) was used to lyse the cells. Cells were incubated for 30 min on ice followed by centrifugation (4 °‍C, 12 000 r/min) for 10 min. The extracted protein was quantified using the bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Waltham, USA). 2.12 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting 2 A handcast gel was prepared with 15% resolving and 6% stacking gel (1%=0.01 g/mL). The lysed proteins were denatured at 95 °C for 5 min, and 15 μg of protein was loaded into each well with 4 μL of Page Ruler™ Prestained Protein Ladder (Thermo Scientific) and resolved at 150 V for 1 h. The proteins were transferred to the polyvinylidene fluoride (PVDF)-trifluoroethylene membrane (GE Healthcare, Amersham, UK) using semi-dry transfer (15 V, 1 h). Membranes were blocked for 2 h with 3% bovine serum albumin (BSA) in Tris-buffered saline with Tween ® 20 (TBST) using gentle agitation. The membrane was then washed (four times for 5 min each), followed by optimized diluted primary antibody incubation. phosphorylated JNK (pJNK) and P38 (pP38) MAPK (Cell Signaling Technology, Danvers, USA) were incubated overnight at 4 °‍C, whereas β‍-actin (Santa Cruz Biotechnology, Santa Cruz, USA) was incubated for 1 h at room temperature. Membranes were then washed followed by secondary antibody conjugated horseradish peroxidase (Santa Cruz Biotechnology) incubation for an hour with gentle agitation. Finally, the membrane was washed and the protein of interest was developed using Western Bright ECL Select (Advansta, San Jose, USA). The signal was captured by the BioSpectrum 600 Imaging System (UVP, Upland, USA). 2.13 Quantification of protein bands 2 The density of protein bands was analyzed using ImageJ quantification software (National Institutes of Health, Sacaton, USA). The relative density of bands was normalized against the respective β-actin relative density. The sample to control density ratio percentage was calculated as per the formula below: density ratio percentage=(normalized sample/normalized control)×100%. 2.14 Statistical analysis 2 Data processing for statistical analysis was performed for cell cycle, apoptosis, and western blotting results. Data are presented as mean±standard deviation (SD) of three independent experiments. Means were compared by one-way analysis of variance (ANOVA) and post-hoc least significant difference (LSD) tests using SPSS Statistics for Windows, Version 20.0 (IBM Corp., Armonk, USA). In each analysis, HC- and 5-FU-treated cells were compared against NT cells. Data are considered to be significant when P < 0.05. 3 Results 1 3.1 Isolation of HC from crude PBL water extract 2 The sample collected was confirmed as Piper betle Linn. by the Medicinal and Aromatic Plant Department at FRIM. Isolated HC was initially verified and quantified using HPLC (Figs. S1 and S2). Based on the equation of the standard curve, the concentration of isolated HC was determined as 1125 ppm (1.125×10 6 μg/mL). Subsequently, NMR and GCMS confirmed the isolated product as HC (Table S1, Figs. S3 and S4). 3.2 Greater cytotoxic effect of HC on HT-29 cells than crude PBL water extract 2 To investigate the potential cytotoxic effect of crude PBL water extract and HC on HT-29 cells, proliferation analysis via MTS assay of these treatments was conducted at various time points with results compared to the standard drug, 5-FU. The latter exerts its cytotoxic effect on HT-29 cells after 72 h of treatment, with IC 50 determined at 50 µmol/L ( Fig. 1 Fig. 1 a). The cytotoxic effect of 5-FU did not continue to rise with increasing 5-FU concentration ( > 50 µmol/L). Crude PBL water extract and HC demonstrated cytotoxic effect on HT-29 cells as early as 24 h (Figs. 1 1 b and 1 1 c). The IC 50 values of PBL extract were determined at 380 µg/mL (24 h) and 220 μg/mL (48 h), respectively ( Fig. 1 Fig. 1 b). The IC 50 of HC was found to be lower concentration compared to the PBL extract, namely, 30 μg/mL at 24 and 48 h ( Fig. 1 Fig. 1 c). Crude PBL water extract and HC exert their cytotoxic effect in a time- and dose-dependent manner. Since the IC 50 values for both these treatments were determined earlier at 24 and 48 h, respectively, the cytotoxic effects of crude PBL water extract or HC at 72 h were not investigated. 1,2,3; 3.3 HC induced G 0 /G 1 cell cycle arrest in HT-29 cells 2 The effects of 5-FU and HC on the cell cycle of HT-29 cells were established by cell cycle analysis using a flow cytometer. The 50 µmol/L treatment of 5-FU induced cell cycle arrest at the G 1 /S phase from 12 to 30 h, which was deduced by the accumulation of cells at the S phase and the decreased population of cells in the G 2 /M phase ( Fig. 2 Fig. 2 ). Treatment with HC (30 µg/mL) induced a G 0 /G 1 phase cell cycle arrest from 12 to 24 h ( Fig. 2 Fig. 2 ). This was evidenced by the accumulation of cell population at the G 0 /G 1 phase and a decline in cell population at the S phase when compared to the NT cells. Interestingly, there was also a significant increase of cell population at the G 2 /M phase at 12 h of HC treatment when compared to the NT cells. Cell cycle arrest was observed at the G 0 /G 1 and also the G 2 /M phases at 12 h of HC treatment. The G 2 /M phase cell population decreased at 18 and 24 h when compared to the NT cells, which implies a lack of cell cycle progression. However, there was a significant ( P < 0.05) decrease in the G 0 /G 1 cell population, an increase in number of cells in the S phase, and a decrease in the G 2 /M phase cell population at 30 h. 3.4 Apoptotic cell death induced by HC as early as 12 h after treatment 2 Flow cytometry analysis by Annexin V-FITC/PI staining enabled the study of the mode of cell death; apoptosis and necrosis were induced by 5-FU and HC. The time-dependent study allowed cell populations to be analyzed over a period of time, observing their progression leading to cell death. Treatment by HC of HT-29 cells led to significant cell death from 12 to 30 h in comparison to the NT cells ( Fig. 3 Fig. 3 ). The apoptotic effect of HC were more prominent than apoptotic cell death triggered by 5-FU treatment. It began to induce apoptosis significantly after 18 h of 5-FU treatment. Nonetheless, a significant increase in cell death through necrosis was observed for HC-treated cells from 24 h onwards in comparison to the NT cells. 1,2;3,4; 3.5 Activation of JNK and P38 MAPK in HT-29 cells after HC treatment 2 The expression of pJNK and pP38 MAPK protein in 5-FU- and HC-treated HT-29 cells was studied using western blot analysis at 12 and 18 h. There was a significant ( P < 0.05) increase in pJNK and pP38 MAPK in HC-treated HT-29 cells at 12 h ( Fig. 4 Fig. 4 ). However, no significant difference was observed in the expression of pJNK or pP38 between NT and 5-FU-treated HT-29 cells at 12 or 18 h. 4 Discussion 1 The TP53 gene is a classical tumor suppressor. In response to cellular stress, such as DNA damage, JNK and P38 regulate cell proliferation and apoptosis through the activation of TP53 . Activated TP53 promotes cell growth arrest, DNA repair, and apoptosis. Cells of the HT-29 line bear TP53 mutation ( Sui et al., 2014 Sui et al., 2014 ) and display chemoresistant features. Mutant TP53 possesses dominant-negative inhibition toward wild-type TP53 . However, the intracellular balance between the expression levels of wild-type and mutant TP53 may play a role in determining cell fate in response to DNA damage ( Ozaki and Nakagawara, 2011 Ozaki and Nakagawara, 2011 ). Our results showed that 5-FU treatment induced G 1 /S cell cycle arrest and a late onset (72 h) of cell death. These observations could be explained by the 5-FU mechanism and the TP53 mutation of HT-29 cells, respectively. 5-FU is a cell cycle-specific, S phase-dependent fluorinated pyrimidine analogue that interferes with thymidylate synthesis. It inhibits thymidylate synthase (TS) in a TP53 -dependent manner ( Sui et al., 2014 Sui et al., 2014 ). Its metabolite, 5-fluorouridine 5'-triphosphate (FUTP), is extensively incorporated into RNA disrupting normal pyrimidine synthesis, subsequently leading to delayed cell cycle progression ( Zhang et al., 2008 Zhang et al., 2008 ). This causes cells to exhibit prolonged DNA synthesis (S phase), and the cells are hence arrested at the G 1 /S phase. Cells with unrepairable damage will then proceed to programmed cell death, i.e., apoptosis. Apoptosis induced by 5-FU is controlled by the TP53 protein; the mutation of TP53 gene is associated with 5-FU resistance due to a reduction in pro-apoptotic protein expression ( Boyer et al., 2004 Boyer et al., 2004 ). In addition, a repression effect of TP53 on pyrimidine catabolic enzyme, dihydropyrimidine dehydrogenase (DPD), has also been reported ( Gokare et al., 2017 Gokare et al., 2017 ). Functional TP53 enables the inhibition of both pyrimidine synthesis and 5-FU catabolism. In the treatment of 5-FU, the lack of functional TP53 is highly undesirable. Cell death triggered by 5-FU was observed in this study, although this occurred at late onset after 72 h of treatment. We compared the cytotoxic effect of PBL and HC to that of 5-FU, and found that PBL water extract and HC treatment exhibited cytotoxic effect on HT-29 cells at 24 h, which is earlier than the cytotoxic effect exerted by 5-FU (72 h). The cytotoxic effect of HC was more potent than that of PBL water extract, which was evident from the lower IC 50 value for HC. Many studies reported that active compounds are more potent than crude extracts in inhibiting cancer cell growth. One such study demonstrated that HC has a greater effect than crude PBL water extract on HT-29 cells ( Ng et al., 2014 Ng et al., 2014 ). Bio-guided fractioning revealed that the fraction containing about 95% of total HC, in comparison to other fractions of PBL extract, exerted the highest cytotoxic effect on prostate cancer cells ( Paranjpe et al., 2013 Paranjpe et al., 2013 ). Polyphenols such as curcumin, gingerols, and capsaicin are active compounds from turmeric, ginger, and red chili, respectively, and these compounds have entered clinical trials as prospective chemotherapeutic agents ( Sung et al., 2012 Sung et al., 2012 ). We also analyzed and compared the cell cycle and apoptotic profiles of 5-FU and HC in a time-dependent manner. As early as 12 h after treatment, HC triggered cell cycle arrest primarily at the G 0 /G 1 and G 2 /M phases, which subsequently led to significantly higher apoptotic cell death of HT-29 cells when compared to 5-FU treatment. This result is in accordance with past studies, where HC-induced apoptosis was observed in consequence of G 0 /G 1 ( Gundala et al., 2014 Gundala et al., 2014 ) and G 2 /M arrest ( Chakraborty et al., 2012 Chakraborty et al., 2012 ; Majumdar and Subramanian, 2019 Majumdar and Subramanian, 2019 ). At 12 h of HC treatment, we found HC-induced phosphorylation of JNK and P38, on which 5-FU did not have any effect. HC has been reported as one of the ROS-induced natural products ( Chakraborty et al., 2012 Chakraborty et al., 2012 ). The JNK and P38 MAPK proteins are expressed in response to various stress stimuli. Induction by oxidative stress activates these proteins, and subsequently leads to the initiation of signaling cascades. In response to oxidative stress, cells can either repair their damage, or cells with irrepairable damage proceed to apoptosis. Therefore, the cell cycle arrest triggered by HC may play an essential role in contributing to the induction of apoptosis. Both JNK and P38 MAPK are involved in apoptosis through the activation of TP53 or B cell lymphoma-2 (Bcl-2) family proteins ( Dhanasekaran and Reddy, 2008 Dhanasekaran and Reddy, 2008 ; Thornton and Rincon, 2009 Thornton and Rincon, 2009 ). Upon activation by P38, phosphorylated TP53 regulates both the G 2 /M and G 1 /S cell cycle checkpoints through the negative regulation of cell division cycle protein 2 (CDC2) and cyclin-dependent kinase 2 (CDK2), respectively ( Thornton and Rincon, 2009 Thornton and Rincon, 2009 ). The JNK regulates TP53 stability ( Dhillon et al., 2007 Dhillon et al., 2007 ) and is involved in (ERK1/2) activation, which subsequently leads to apoptosis induction ( Chowdhury et al., 2013 Chowdhury et al., 2013 ). It also phosphorylates c-Jun, which then leads to the formation of activator protein-1 (AP-1). The JNK-AP-1 pathway induces the expression of pro-apoptotic genes such as tumor necrosis factor-α ( TNF-‍α ), Fas-ligand ( Fas-L ), and Bcl-2 homologous antagonist/killer ( Bak ) ( Dhanasekaran and Reddy, 2008 Dhanasekaran and Reddy, 2008 ). However, HT-29 used in our study possesses mutant TP53 . Our results evidenced that mutant TP53 did not suppress HC-induced apoptosis. Both JNK and P38 activation are essential for apoptotic response to HC-induced cell death, and may be independent of TP53 activity. Nevertheless, a reactivation of mutant TP53 is possible and provide clues in killing cancer cells bearing the TP53 mutation. Several small molecules ( Wiman, 2010 Wiman, 2010 ; Brown et al., 2011 Brown et al., 2011 ; Zawacka-Pankau and Selivanova, 2015 Zawacka-Pankau and Selivanova, 2015 ) and natural products ( Kitagishi et al., 2012 Kitagishi et al., 2012 ; Garufi et al., 2016 Garufi et al., 2016 ) have been tested for TP53 reactivation in mutant TP53 -carrying cells. A study by Garufi et al. (2016) Garufi et al. (2016) found that capsaicin, the major constituent of peppers, reduces mutant TP53 levels and reactivates the wild-type TP53 protein in mutant TP53 -carrying cells. The clearance of TP53 mutation has been reported to reduce tumor malignancy and increase the apoptotic response to drugs ( Bossi et al., 2006 Bossi et al., 2006 ; Martins et al., 2006 Martins et al., 2006 ). Taken together, we have demonstrated that HC possesses more potent cytotoxic effect than crude PBL water extract or 5-FU on HT-29 cells. Our study provides information on the possible involvement of JNK and P38 MAPK in HC-induced cell cycle arrest and apoptotic cell death of HT-29 cells, which may be independent of TP53 activity. Nevertheless, the reactivation of mutant TP53 is still possible. These findings have given more insight into the potential mechanism of action of HC. References Abrahim NN , Kanthimathi MS , Abdul-Aziz A , 2012 . 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Mee Lee LOOI and Aiysvariyah RAJEDADRAM contributed to the writing and editing of the manuscript. All authors have read and approved the final manuscript and, therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. This reasearch was supported by the Taylor’s Research Grant Scheme (No. TRGS/MFS/2/2013/SBS/003), Malaysia.We thank Taylor’s University and Forest Research Institute Malaysia (FRIM) for the laboratory facilities. 10.1631/jzus.B2000446 1673-1581（2021）02-0112-11 RAJEDADRAM Aiysvariyah,PIN Kar Yong,LING Sui Kiong et al., 2021. Hydroxychavicol, a polyphenol from Piper betle leaf extract, induces cell cycle arrest and apoptosis in TP53 -resistant HT-29 colon cancer cells. Journal of Zhejiang University-SCIENCE B(Biomedicine & Biotechnology), 22(2):112-122. RAJEDADRAM Aiysvariyah,PIN Kar Yong,LING Sui Kiong et al., 2021. Hydroxychavicol, a polyphenol from Piper betle leaf extract, induces cell cycle arrest and apoptosis in TP53-resistant HT-29 colon cancer cells. Journal of Zhejiang University-SCIENCE B(Biomedicine & Biotechnology), 22(2):112-122. 2020-08-07 2021-01-06 2021-02-15 2022-05-09 © Zhejiang University Press 2021 Aiysvariyah RAJEDADRAM, Kar Yong PIN, Sui Kiong LING, See Wan YAN, and Mee Lee LOOI declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. Compliance with ethics guidelines Supplementary information 1 Table S1; Figs. S1‒S4 浙江大学学报（英文版）（B辑：生物医学和生物技术） 2 22