{"id":51874,"date":"2023-09-30T11:10:52","date_gmt":"2023-09-30T11:10:52","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=51874"},"modified":"2023-10-07T09:16:57","modified_gmt":"2023-10-07T09:16:57","slug":"chemopreventive-measurements-and-oxidative-stress-effects-of-terpenoid-rich-canarium-odontophyllum-miq-leaf-extract-trco-in-ultraviolet-b-induced-in-vitro-skin-carcinogenesis-model","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol16no3\/chemopreventive-measurements-and-oxidative-stress-effects-of-terpenoid-rich-canarium-odontophyllum-miq-leaf-extract-trco-in-ultraviolet-b-induced-in-vitro-skin-carcinogenesis-model\/","title":{"rendered":"Chemopreventive Measurements and Oxidative Stress Effects of Terpenoid-rich Canarium odontophyllum Miq. Leaf Extract (TRCO) in Ultraviolet B-Induced In-Vitro Skin Carcinogenesis Model"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

The most frequently occurring cancer in humans is non-melanoma skin cancer, also known as keratinocyte carcinoma. This cancer usually appears on skin areas exposed to the sun, including the head, neck, shoulders, back, and arms. These cancers have recently increased significantly worldwide.1<\/sup> Solar ultraviolet radiation, especially ultraviolet-B (UVB) is a significant environmental carcinogen and the main culprit for human skin cancer.2<\/sup> UVB exerts more negative impact than beneficial to living organisms. UVB radiation triggers the skin to produce free radicals or reactive oxygen species (ROS), which have a significant role in the disease’s development. Consecutively, antioxidants are essential for safeguarding our body from free radicals’ damaging effects.3<\/sup> However, the body’s inherent antioxidant defence mechanism has certain limitations,4<\/sup> and can be overwhelmed by exposure to excessive ultraviolet (UV) radiation. Thus, taking additional measures to safeguard the skin from damage caused by sunlight and maintaining cellular redox balance is crucial. Applying exogenous antioxidants on the skin’s surface can raise antioxidant levels in the human epidermis, protecting against oxidative damage caused by UVB radiation.5<\/sup> In plants’ defence system, terpenoid is an example of their photoprotective molecule. Terpenoids which are a wide group of secondary plant metabolites have shown significant potential as chemopreventive and therapeutic agents in cancer treatment.6<\/sup> Recently, there has been growing attention towards discovering novel external antioxidants from plant sources in mitigating oxidative stress-related ailments that offer photoprotective benefits. Natural compounds are considered safer and more promising substitutes for commonly used synthetic antioxidants.7<\/sup> <\/p>\n\n\n\n\n\n
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Figure 1: Canarium odontophyllum<\/em> Miq. (Dabai) tree<\/strong><\/p>\n

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Canarium odontophyllum<\/em> Miq., also recognised as the \u201cdabai\u201d fruit, is particularly rich in antioxidants. The natives in Sarawak, Malaysia, consume C. odontophyllum<\/em> Miq., as a snack to support their health and vitality.8<\/sup> The tree (Figure 1) can reach a height of 36 m and a diameter of 85 cm.9<\/sup> It also has oblong leaves with 3\u20138 pairs of spirally arranged leaflets, with the terminal leaflets at the end.10<\/sup> The edible fruits are fondly known as the ‘Sibu olives’. The fruit contains thin, dark, purple-coloured skin (epidermis), thick golden-coloured flesh (mesocarp) and a single seed (endocarp).<\/p>\n\n\n\n

To date, there has been limited research on the biological characteristics of Canarium odontophyllum<\/em> Miq. There have been claims of medicinal use of the C. odontophyllum<\/em> Miq. leaf by locals in Sarawak. Previous investigations have examined various biological activities of C. odontophyllum<\/em> Miq. leaf extracts.11<\/sup> Based on our previous C. odontophyllum<\/em> leaf phytochemical profiling, we determined the active compounds found in nonpolar organic solvents, particularly hexane, using gas chromatography-mass spectrometry (GC-MS). The extract obtained is rich with terpenoids, including major terpenoids such as spathulenol and phytol, and primary fatty acid (palmitic acid) that might be responsible for their biological activities.12<\/sup>  Fatty acids are recognised for acting as antioxidants and scavenging free radicals.13<\/sup> Furthermore, terpenoids exhibit several properties, including antimicrobial, antioxidant, anti-inflammatory, anti-malarial, anti-cancer, and anti-fungal activities.14<\/sup><\/p>\n\n\n\n

Hence,\nour investigation aimed to determine the chemopreventive measurements and\noxidative stress effects of terpenoids-rich C. odontophyllum<\/em> Miq. leaf\nextract (TRCO) on chronic UVB-induced in-vitro skin damage.<\/p>\n\n\n\n

Materials\nand methods<\/strong><\/p>\n\n\n\n

Studied Plant and TRCO preparation<\/strong><\/p>\n\n\n\n\n\n
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Figure 2: Canarium odontophyllum<\/em> Miq. (Dabai) leaves<\/strong><\/p>\n

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Fresh leaves of C. odontophyllum<\/em> Miq. (Figure 2) were gathered from Kuching, Sarawak, Malaysia, with permits acquired from the Sarawak Biodiversity Centre (SBC-2020-EP-58-MWH & SBC-2019-RDP-20-MWH). The leaf sample was deposited in the herbarium (Universiti Kebangsaan Malaysia (UKM)) with voucher number ID028\/2020. The process to obtain a terpenoid-rich extract yielded 0.499 g (0.624%), and TRCO test concentrations (500 \u00b5g\/ml TRCO (TRCO500) & 1000 \u00b5g\/ml TRCO (TRCO1000)) followed the procedure from our previous study.12<\/sup> <\/p>\n\n\n\n

UVB-induced Immortalized Human\nKeratinocytes (HaCaT)<\/strong><\/p>\n\n\n\n

Prolonged UVB Irradiation<\/strong><\/p>\n\n\n\n

UVB source was produced from UVB Lamps (Analytikjena USA), and the intensity was measured using a UVX digital radiometer (Analytikjena USA). The lamps were switched on for at least 5 minutes before the dose rate of UVB, which was 30 mJ\/cm2<\/sup>, was measured.15<\/sup><\/p>\n\n\n\n

Immortalized HaCaT<\/strong><\/p>\n\n\n\n

The HaCaT cell line vial (EP-CL-0090) was purchased from Elabscience (USA). The cell revival, subculture and calculation followed the detailed procedures in the previous study.12<\/sup> The 6-well cell culture plates were used to culture the cells. The wells in plates were first added with 2 ml of DMEM media. Then, cells were seeded in 6-well plates at a density of 4 x 105<\/sup> cells per well. The media was changed every 48 hrs and were checked for 80% confluency.<\/p>\n\n\n\n

HaCaT oncogenic transformation model<\/strong><\/p>\n\n\n\n

In-vitro UVB-induced skin carcinogenesis model from a previous study15<\/sup> was adapted with optimisations. Upon reaching 80% cell confluency during passage (P) 5 (P5), media was removed from wells in the 6-well plates. Cells were then given two PBS washes and added 1 ml of media into each well. Following that, the plates containing the cells were incubated for thirty minutes at 37\u00b0C with 5% CO2<\/sub>. After incubation, The UVB-induced HaCaT group was exposed to 30 mJ\/cm2 <\/sup>UVB radiation. The non-UVB-induced group was devoid of UVB exposure. Following receiving UVB radiation, the cells were given a new medium, and they were then incubated at 37\u00b0C with 5% CO2<\/sub> until confluent. This method was repeated for the next P6 until P10 (6 passages).<\/p>\n\n\n\n

Giemsa Staining<\/strong><\/p>\n\n\n\n

Standard\nGiemsa staining was done to verify the optimised model of HaCaT transformation.\nIt aided the visualisation to observe the cells\u2019 morphological changes, such as\nthe shape and size of the cells and their nuclei. The adhered cells in 6-well\nplates (P11) were first fixed for 5 minutes in ethanol and then air-dried.\nThen, the Giemsa stain solution (Sigma-Aldrich, USA) was added to each well and\nallowed to sit for 20 minutes. After staining, the wells were washed with water\nand air-dried before viewing under a microscope.<\/p>\n\n\n\n

Cell\nLysates Preparation<\/strong><\/p>\n\n\n\n

The\noncogenic transformation model was carried out for TRCO groups. TRCO500 and\nTRCO1000 were added into 1 ml media before UVB radiation from P5-P10. After\nreaching P11, each of the wells containing HaCaT cells from all treatment\ngroups (non-UVB, UVB-induced, UVB + TRCO500 & UVB + TRCO1000) was added 150\n\u00b5l of ice-cold RIPA lysis solution, which afterwards incubated for five minutes\non ice. Each well was gently scraped after incubation using a cell scraper to\nremove and lyse cells. The cells were then put into cool micro-centrifuge\ntubes. After that, cells underwent a 30-minute incubation on ice with continual\nagitation. The cells were centrifuged at 4\u00b0C for 10 minutes at 12000 rpm. The\nsupernatant was carefully transferred (prevented the pellet) to a new\nmicrocentrifuge tube and then kept at -80\u00b0C for total protein count (BSA)\n(Elabscience, USA, Catalog No. E-BC-K318-M) and further tests.<\/p>\n\n\n\n

Chemopreventive\nMeasurements<\/strong><\/p>\n\n\n\n

Human marker of proliferation KI-67 (KI67) Content Assay<\/strong><\/p>\n\n\n\n

The\nKI67 content in treatment groups was determined following the human MKI67\n(Antigen KI-67) ELISA kit (Catalog No. EH0684) from the FineTest, China.\nSamples (n=9) and standards were prepped per the manufacturer’s manual in the\nkit. 100 \u00b5L of diluted samples were added to 96 well plates for assay\nprocedures, and OD 450 nm was measured using a microplate reader (Multiskan Go,\nThermo Scientific, USA). The examined samples’ KI67 was then determined using\nthe standard curve obtained.  <\/p>\n\n\n\n

Human Tumour Protein p53 (TP53) Content Assay<\/strong><\/p>\n\n\n\n

The\nTP53 content in treatment groups was determined following the human total TP53\nELISA kit (Catalog No. EH3898) from FineTest, China. Samples (n=9) and\nstandards were prepped per the manufacturer’s manual in the kit. 100 \u00b5L of\ndiluted samples were added to 96-well plates for assay procedures and then\nmeasured at OD 450 nm using a microplate reader (Multiskan Go, Thermo\nScientific, USA). The TP53 in the tested samples was then calculated using the\nestablished standard curve.<\/p>\n\n\n\n

Determination of Biochemical Markers for Oxidative\nStress Effects<\/strong> <\/p>\n\n\n\n

Total Superoxide Dismutase (T-SOD) Activity<\/strong><\/p>\n\n\n\n

According to the T-SOD activity test kit (Catalog No. E-BC-K020-M) from Elabscience, USA, the T-SOD activity in treatment groups was measured. Samples (n=9) were prepared following the kit’s manufacturer’s protocol. The enzyme working solution and the samples, each 20 \u00b5l in volume, were put into a 96-well plate. Each well was then filled with 200 \u00b5l of the substrate application solution and properly mixed. Once the plate had been incubated at 37\u00b0C for 20 minutes, the OD values at 450 nm were measured using a microplate reader (Multiskan Go, Thermo Scientific, USA). The T-SOD activity in the samples was then determined using the formula below:<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

where,<\/p>\n\n\n\n

i <\/em> : Inhibition ratio of SOD (%)<\/p>\n\n\n\n

V1: The\ntotal volume of the reaction, 240 \u00b5l<\/p>\n\n\n\n

V2: The volume of sample added to the\nreaction, 20 \u00b5l<\/p>\n\n\n\n

f<\/em> : Dilution factor of the sample before tested<\/p>\n\n\n\n

Cpr<\/em> : Protein concentration of sample, mgprot\/ml<\/p>\n\n\n\n

T-SOD activity for each treatment group and\ncontrols were calculated and recorded as mean \u00b1 SEM.<\/p>\n\n\n\n

Catalase (CAT) Activity Assay<\/strong><\/p>\n\n\n\n

The\nCAT activity in treatment groups was measured following the CAT activity assay\nkit (Catalog No. E-BC-K031-S) from Elabscience, USA. Samples (n=9) were\nprepared per the manufacturer’s manual in the kit. Then, 100 \u00b5l of samples were\ntransferred to 96-well plates for OD 405 nm measurement with a microplate\nreader (Multiskan Go, Thermo Scientific, USA). The following formula was used\nto determine the quantity of catalase present in the tested samples:<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

where,<\/p>\n\n\n\n

32.5: reciprocal of the slope<\/p>\n\n\n\n

1: reaction time<\/p>\n\n\n\n

DA: Absolute OD (ODControl<\/sub> \u2013 ODSample<\/sub>)<\/p>\n\n\n\n

V: Volume of sample, ml<\/p>\n\n\n\n

f <\/em>: Dilution factor of the sample before the test<\/p>\n\n\n\n

Cpr<\/em>: Concentration of protein in the sample, gprot\/L<\/p>\n\n\n\n

CAT activity for each treatment group and\ncontrols were calculated and recorded as mean \u00b1 SEM.  <\/p>\n\n\n\n

Glutathione peroxidases (GSH-Px) Activity<\/strong><\/p>\n\n\n\n

GSH-Px\nactivity in treatment groups was measured following the GSH-Px activity assay\nkit (Catalog No. E-BC-K096-S) from Elabscience, USA. Samples (n=9) were\nprepared per the manufacturer’s protocol in the kit. Two sets of reactions\n(enzymatic and non-enzymatic) were prepared for each treatment group. A glutathione\n(GSH) standard was prepared. The GSH-Px activity determination assay involved\ntwo sections; i) enzymatic reaction and ii) chromogenic reaction, carried out\nper the kit’s protocol. Then, 100 \u00b5l of samples were added to 96-well plates\nfor OD measurement at 412 nm using a microplate reader (Multiskan Go, Thermo\nScientific, USA). GSH-Px activity in samples was then calculated according to\nthe formula below:<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

where,<\/p>\n\n\n\n

\ud835\udf1fA1: OD non-enzyme\ntube – OD enzyme tube<\/p>\n\n\n\n

\ud835\udf1fA2: OD Standard – OD\nBlank  <\/p>\n\n\n\n

c: concentration of standard, 20 \u00b5mol\/L <\/p>\n\n\n\n

d: the optical path of 96 well plates, 0.28cm <\/p>\n\n\n\n

V: volume of sample added to the reaction\nsystem, ml <\/p>\n\n\n\n

f: dilution factor of the sample before the\ntest <\/p>\n\n\n\n

f2: dilution factor of cell culture in\nenzymatic reaction, 5 times <\/p>\n\n\n\n

Cpr: Concentration of protein in the sample<\/p>\n\n\n\n

GSH-Px activity for\neach treatment group and controls were calculated and recorded as mean \u00b1 SEM.<\/p>\n\n\n\n

Glutathione-S-transferase (GST) Activity<\/strong><\/p>\n\n\n\n

GST\nactivity in treatment groups was determined following the GST activity assay\nkit (Catalog No. E-BC-K278-S) from Elabscience, USA. Reagents and samples (n=9)\nwere prepared, and the assay was carried out per the manufacturer’s protocol in\nthe kit. Then, 100 \u00b5l of samples were added to 96-well plates. Next, OD\nmeasurements were obtained at two specific times: i) 20 s and ii) 320 s at 340\nnm using a microplate reader (Multiskan Go, Thermo Scientific, USA). The\nfollowing formula was used to determine the GST activity in the samples:<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

where,<\/p>\n\n\n\n

\ud835\udf1fA : OD sample – OD blank <\/p>\n\n\n\n

\u03a3 : molar extinction coefficient of the product, 9.6 X 103<\/sup> L\/mol\/cm <\/p>\n\n\n\n

d: the optical path of 96 well plates, 0.28cm <\/p>\n\n\n\n

106<\/sup>: 1 mol= 106 <\/sup>\u00b5mol <\/p>\n\n\n\n

V1: total volume of the reaction system, 1.1ml\n<\/p>\n\n\n\n

V2: volume of sample added to the reaction\nsystem, 0.1ml <\/p>\n\n\n\n

t: reaction time, 5 min <\/p>\n\n\n\n

f: dilution factor of the sample before the\ntest <\/p>\n\n\n\n

Cpr: Concentration of protein in the sample<\/p>\n\n\n\n

GST activity for\neach treatment group and controls were calculated and recorded as mean \u00b1 SEM.<\/p>\n\n\n\n

Lipid peroxide (LPO) Content<\/strong><\/p>\n\n\n\n

The LPO content determination in the treatment groups\nwas done following the manufacturer’s instruction by using LPO colourimetric\nassay kit (Catalog No: E-BC-K176-M) from Elabscience, USA. Firstly, a standard\ncurve of different concentrations ranging from 0, 5, 10, 20, 30, 40, 50, and 80\n\u00b5mol\/L was prepared using 100 \u00b5mol\/L standard solutions in the kit. The linear\nequation y=ax+b is obtained by plotting a standard curve using the OD value of\nstandards (y-axis) and its matching concentration (x-axis). Next, 200 \u00b5l of\neach sample (n=9) was prepared following the kit’s instructions. Then, the colourimetric\nchanges in samples indicating the presence of LPO were measured using a\nmicroplate reader (Multiskan Go, Thermo Scientific, USA) at OD 562 nm. The\nconcentration of LPO in the tested samples was then calculated using the\nformula below based on the plotted standard curve:<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

where,\n<\/p>\n\n\n\n

\u0394A562:\nOD sample- OD Blank<\/p>\n\n\n\n

a:\nslope of the standard curve <\/p>\n\n\n\n

b:\nintercept of the standard curve <\/p>\n\n\n\n

f:\ndilution factor of the sample before the test <\/p>\n\n\n\n

Cpr:\nConcentration of protein in the sample<\/p>\n\n\n\n

The\nLPO content for each treatment group and control was calculated and recorded as\nmean \u00b1 SEM.<\/p>\n\n\n\n

Protein carbonyl (PC) Content<\/strong><\/p>\n\n\n\n

According\nto the PC colourimetric assay kit (Catalog No. E-BC-K117-S from Elabscience,\nUSA), the protein carbonyl concentration of treatment groups was measured.\nSamples (n=9) were prepped per the manufacturer’s manual in the kit. Then, 100 \u00b5L\nof samples were transferred to 96-well plates for measurement of OD at 370 nm\nusing a microplate reader (Multiskan Go, Thermo Scientific, USA). The formula\nprovided below was utilised to calculate the concentration of PC in the tested\nsamples:  <\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

where,<\/p>\n\n\n\n

A1: OD value of the sample <\/p>\n\n\n\n

A2: OD value of control <\/p>\n\n\n\n

\u03a3 : molar extinction coefficient of carbonyl, 22000 L\/mol\/cm <\/p>\n\n\n\n

d: the optical path of 96 well plates, 0.28cm <\/p>\n\n\n\n

V1: total volume of the reaction system,\n1.25ml <\/p>\n\n\n\n

V2: total volume of sample added to the reaction system, 0.1ml <\/p>\n\n\n\n

106<\/sup>: unit conversion, 1 mol= 109<\/sup> nmol <\/p>\n\n\n\n

f: dilution factor of the sample before the\ntest <\/p>\n\n\n\n

Cpr: Concentration of protein in the sample<\/p>\n\n\n\n

PC content for each treatment group and\ncontrols were calculated and recorded as mean \u00b1 SEM.  <\/p>\n\n\n\n

Statistical Analysis<\/strong><\/p>\n\n\n\n

GraphPad\nPrism Version 9.3.1 was employed for statistical analysis. All experiments were\ndone in triplicate (n=3), and the findings were presented as means \u00b1 standard\nerror of mean (SEM). The means of several treatment groups were compared using\na one-way ANOVA test with post-hoc analysis. A significance level of 0.05 has\nbeen established, meaning that results with a p-value less than 0.05\n(p<0.05) will be deemed statistically significant.<\/p>\n\n\n\n

Results\nand Discussion<\/strong><\/p>\n\n\n\n

In-vitro UVB-induced HaCaT Oncogenic\nTransformation<\/strong><\/p>\n\n\n\n\n\n
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Figure 3: Morphology of HaCaT cells (a) <\/strong>non-UVB induced (b) UVB induced (30 mJ\/cm2<\/sup> x 6 passages (P5-P10)) on magnifications 10x & 40x. Giemsa. PM: Pleomorphism<\/strong><\/p>\n

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Giemsa-stained cells appeared to be pink to purple, while the nuclei appeared blue to purple. Results showed observable oncogenic transformation in cell morphology caused by repeated UVB radiation. Non-UVB-induced HaCaT cells showed normal morphology of small, uniformly shaped nuclei and conformity in cell size and shape (Figure 3(a)); meanwhile, UVB-induced HaCaT cells showed pleomorphism and disorganised arrangement (Figure 3(b)). Previous studies that have been published claim that a minimal erythemal dose of UVB of about 40 mJ\/cm2<\/sup> 16<\/sup> results in DNA damage and substantial apoptosis in radiated keratinocytes.17<\/sup> Hence, to depict a realistic scenario of recurring DNA damage and repair, we selected a sub-erythemal dose of UVB (30 mJ\/cm2<\/sup>), which enabled HaCaT to be exposed to it repeatedly.<\/p>\n\n\n\n

Chemopreventive\nMeasurements<\/strong><\/p>\n\n\n\n

Human KI67 Content<\/strong><\/p>\n\n\n\n\n\n
\"\"<\/td>\nGraph 1: <\/strong>Effect of TRCO on the KI67 content in HaCaT cell treatment
groups. Values represent mean \u00b1 SEM of 3 independent experiments. <\/strong>

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Pretreated groups TRCO500 & TRCO1000 significantly reduced the expression of KI67 in cells (91.54 \u00b1 1.32 ng\/ml and 83.75 \u00b1 2.55 ng\/ml, respectively) compared to UVB induced group (105.31 \u00b1 0.92 ng\/ml) (Graph 1). KI67 protein was a marker found in cells and was highly linked to cell proliferation.  The KI67 antigen existed solely in the nucleus of cells during the interphase stage, but during mitosis, a significant portion of the protein moved to the chromosomal surface.  The KI67 protein can be found in the cell cycle’s active stages, which include G1, S, G2, and mitosis. However, the protein was absent in inactive or quiescent cells (G0).18<\/sup> A study found that the quantity of KI67 protein in cells increased as they progressed through the cell cycle’s S phase.19<\/sup> Based on the findings, it was shown that the TRCO has some role in regulating KI67. Terpenoids such as spathulenol, which can be found in abundance in TRCO might contribute as an anti-proliferative agent, which spathulenol has been reported previously to have anti-proliferative properties.20<\/sup><\/p>\n\n\n\n

Human TP53 Content<\/strong><\/p>\n\n\n\n\n\n
\"\"<\/td>\nGraph 2: <\/strong>Effect of TRCO on the tumour P53 content in HaCaT cell treatment groups. Values represent mean \u00b1 SEM of 3 independent experiments. <\/strong>

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Our findings showed the pretreated group TRCO1000 (76.09 \u00b1 2.11 ug\/ml) significantly reduced TP53 expression compared to the UVB-induced group (84.68 \u00b1 2.34 ug\/ml) (Graph 2). However, the pretreated group with TRCO500 showed no decrease in TP53 expression. This result might be due to more proliferated cells than the TRCO1000 group, as stated in Figure 4. The functionality of the p53 gene was compromised by a genetic mutation, resulting in the loss of its ability to promote tumour suppression and thereby initiate tumorigenesis. Whenever there is genotoxic stress, P53 protein levels are increased. DNA damage, hypoxia, and mitogenic oncogenes are a few biological stresses that can activate the p53 gene.21<\/sup> <\/p>\n\n\n\n

Determination of Biochemical Biomarkers of TRCO on UVB-induced HaCaT<\/strong> Carcinogenesis Model<\/strong> <\/p>\n\n\n\n

T-SOD Activity<\/strong><\/p>\n\n\n\n\n\n
\"\"<\/td>\nGraph 3:<\/strong> Effects of TRCO on T-SOD activity in HaCaT cell treatment groups. T-SOD: total superoxide dismutase. Values represent mean \u00b1 SEM of 3 independent experiments.<\/strong>

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Our study demonstrated that HaCaT cells experienced a reduction in T-SOD (total superoxide dismutase) activity due to repeated exposure to UVB (Graph 3). Our results are consistent with the previous research, which has also shown that UVB exposure can result in T-SOD deficiency.22<\/sup>  Pretreatment of 500 \u00b5g\/ml TRCO (TRCO500) effectively shielded cells from UVB radiation’s harmful effects and considerably raised the SOD activity to 32.73 \u00b1 0.16 U\/mgprot. However, for the 1000 \u00b5g\/ml TRCO (TRCO1000) group, there was no significant increase in SOD activity (28.79 \u00b1 0.21 U\/mgprot) compared to the UVB-induced group (27.99 \u00b1 0.32 U\/mgprot). The lack of a significant increase in SOD activity at the higher concentration of TRCO may be due to the combination of UVB exposure and higher cytotoxicity, as shown in the MTT assay reported previously.12<\/sup>  Overall, our results indicate that TRCO may be able to guard skin cells against the damaging effects of persistent UVB exposure.<\/p>\n\n\n\n

CAT Activity<\/strong><\/p>\n\n\n\n\n\n
\"\"<\/td>\nGraph 4:<\/strong> Effects of TRCO on CAT activity in HaCaT cell treatment groups. CAT: catalase.  Values represent mean \u00b1 SEM of 3 independent experiments.<\/strong>

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UVB\nexposure significantly increased catalase activity (Graph 4). Pre-treatment of\nTRCO500 and TRCO1000 seemed to significantly reduce catalase enzyme activity\n(11.63 \u00b1 0.05 U\/mgprot and 18.68 \u00b1 0.25 U\/mgprot, respectively) in UVB-induced\nHaCaT and the trend was similar to T-SOD where TRCO500 showed higher potency\nthan TRCO1000. The ROS generation was shown to be increased in the UVB-induced\nkeratinocytes that overexpressed catalase significantly. Catalase absorbed UVB\nradiation and converts it into reactive chemical intermediates, which can\nneutralise the antioxidant enzymes available inside the cell.23<\/sup><\/a> However, excessive ROS accumulation due to catalase\nactivity can result in oxidative stress and DNA damage, potentially leading to\nskin cancer.24<\/sup><\/a> TRCO successfully inhibited the production of ROS and\nreduced ROS accumulation in the cells.<\/p>\n\n\n\n

GSH-Px Activity<\/strong><\/p>\n\n\n\n\n\n
\"\"<\/td>\nGraph 5:<\/strong> Effects of TRCO on GSH-Px activity in HaCaT cell treatment groups. GSH-Px: glutathione peroxidase. Values represent mean \u00b1 SEM of 3 independent experiments.<\/strong>

<\/p>\n

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TRCO500\nand TRCO1000 groups exhibited pro-oxidative activity (46.88 \u00b1 7.04 U\/mgprot and\n51.29 \u00b1 10.03 U\/mgprot, respectively) compared to the control group (182.79 \u00b1\n14.12 U\/mgprot). However, the UVB exposure significantly depleted the GSH-Px\nactivity (Graph 5). Depletion in GSH-Px was caused by the exhaustion of the said\nenzyme and its substrate GSH to neutralise and reduce UVB-induced free radicals,\nand GSH-Px activity was not unlimited.25<\/sup><\/a> Depletion\nin GSH by the UVB & TRCO may lead to a reduced GSH-Px activity.<\/p>\n\n\n\n

GST Activity<\/strong><\/p>\n\n\n\n\n\n
\"\"<\/td>\nGraph 6:<\/strong> Effects of TRCO on GST activity in HaCaT cell treatment groups. GST: glutathione  s-transferase. Values represent mean \u00b1 SEM of 3 independent experiments. <\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

The\nUVB exposure significantly increased GST activity (Graph 6). High GST activity\nin the UVB group could indicate that the enzyme was triggered due to an\nincrease in free radical production. Pretreatment of TRCO500 and TRCO1000, with\nhigh antioxidative capacity, resulted in an overall reduction (0.00088 \u00b1\n0.000047 U\/mgprot and 0.00044 \u00b1 0.000045 U\/mgprot respectively) in oxidative\nstress compared to UVB-induced group (0.00338 \u00b1 0.000112 U\/mgprot). Our\nfindings may conclude that TRCO acted as antioxidants which helped eliminate\nfree radicals and did not necessitate induction of high GST activity. The\nprimary function of GST was to eliminate electrophiles (free radicals) through\nconjugation with reduced glutathione (GSH) and can also detoxify lipid\nperoxidation (LPO).26<\/sup><\/a> <\/p>\n\n\n\n

Determination\nof oxidative damage markers of TRCO on Chronic UVB-induced HaCaT<\/strong><\/p>\n\n\n\n

LPO Level<\/strong><\/p>\n\n\n\n\n\n
\"\"<\/td>\nGraph 7:<\/strong> Effect of TRCO on LPO level in HaCaT cell treatment groups. LPO: lipid peroxides. Values represent mean \u00b1 SEM of 3 independent experiments. <\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

TRCO500 and TRCO1000 groups showed a decreased LPO formation caused by UVB-induced free radicals (0.34 \u00b1 0.04 \u00b5mol\/gprot and 0.30 \u00b1 0.06 \u00b5mol\/gprot respectively) but not statistically significant compared to UVB-induced group (0.44 \u00b1 0.04 \u00b5mol\/gprot) (Graph 7). The epidermis layer of skin was composed of a myriad of lipids which were targets of free radicals leading to lipid peroxidation.27<\/sup> UVB exposure induced LPO formation.28,29<\/sup> Exogenous antioxidants directly scavenge free radicals, thus, reducing the lipid peroxidation process.30<\/sup> Exogenous antioxidants directly scavenge free radicals, thus, reducing the lipid peroxidation.<\/p>\n\n\n\n

PC Level<\/strong><\/p>\n\n\n\n\n\n
\"\"<\/td>\nGraph 8:<\/strong> Effect of TRCO on PC level in HaCaT cell treatment groups. Values
represent mean \u00b1 SEM of 3 independent experiments. <\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

In this study, we found that groups pretreated with TRCO500 and TRCO1000 significantly reduced the formation of protein carbonyls compared to the UVB-induced group (Graph 8). The level of protein carbonyls in both TRCO pretreated groups was measured at 20.95 \u00b1 1.73 nmol\/mgprot and 19.24 \u00b1 1.92 nmol\/mgprot, respectively, while the level in the UVB-induced group was much higher at 67.24 \u00b1 5.47 nmol\/mgprot. Exposure to UVB rays generates ROS that triggers protein oxidation with a substantial rise in protein carbonyls, an indicator of protein oxidation, due to UVB exposure.31<\/sup> This means that TRCO could potentially prevent the oxidation of proteins caused by UVB radiation by decreasing the generation of protein carbonyls.<\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

Our\nstudy adapted the in-vitro skin carcinogenesis model to evaluate the chemopreventive\nmeasurements of TRCO. Our research findings suggest that TRCO has potential\nbenefits in the chemoprevention of skin cancer induced by UVB radiation.\nSpecifically, TRCO demonstrated chemopreventive effects by acting as an\nanti-proliferative agent through the KI67 pathway and suppressing the TP53.\nTRCO also helped reduce oxidative stress in HaCaT cells caused by repeated UVB\nradiation. In conclusion, TRCO has the potential to be studied further with\nisolations of its terpenoids as a possible therapeutic remedy for skin cancer\nprevention. These findings emphasise the possible advantages of utilising\nnatural plant extracts for preventing and treating skin cancer and lay the\nfoundation for additional research in this domain.<\/p>\n\n\n\n

Acknowledgement<\/strong><\/p>\n\n\n\n

We would like to\nacknowledge Sarawak Biodiversity Centre for the C. odontophyllum<\/em> Miq research\npermit, Universiti Malaysia Sarawak for the emolument and Faculty of Health\nSciences, Universiti Kebangsaan Malaysia for the lab facilities throughout the\nstudy.<\/p>\n\n\n\n

Conflict\nof Interest<\/strong><\/p>\n\n\n\n

All authors declare\nthat there is no conflict of interest.<\/p>\n\n\n\n

Funding\nSource<\/strong><\/p>\n\n\n\n

This project was\nfunded by Universiti Kebangsaan Malaysia under a research code grant\n(DIP-2018-034).<\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

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