J Cancer Prev 2022; 27(4): 221-228
Published online December 30, 2022
© Korean Society of Cancer Prevention
1Core-Facility Center for Tissue Regeneration, Dong-eui University, 2Biopharmaceutical Engineering Major, Division of Applied Bioengineering, College of Engineering, Dong-eui University, 3Department of Biopharmaceutics, Dong-eui University Graduate School, 4Department of Biochemistry, College of Korean Medicine, 5Blue-Bio Industry Regional Innovation Center, Dong-eui University, Busan, Korea
Correspondence to :
Hyun Ju Kwon, E-mail: firstname.lastname@example.org, https://orcid.org/0000-0002-1375-0906
Byung Woo Kim, E-mail: email@example.com, https://orcid.org/0000-0001-7940-1074
*These authors contributed equally to this work as co-first authors.
**These authors contributed equally to this work as co-correspondence authors.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Cedrol, a sesquiterpene alcohol, isolated from Juniperus chinensis has been reported to inhibit minichromosome maintenance (MCM) proteins as cancer biomarkers in human lung cancer in vitro. In the present study, we investigated the anti-cancer activity of cedrol in vitro and in vivo using human colorectal cancer HT29 cells and a human colorectal tumor xenograft model. Cedrol inhibited MCM protein expression and cell growth in HT29 cells, which are associated with G1 arrest and the induction of apoptosis. We demonstrated that cedrol effectively reduced HT29 tumor growth without apparent weight loss in a human tumor xenograft model. Compared with vehicle- and adriamycin-treated tumor tissues, cedrol induced changes in the tumor tissue structure, resulting in a reduced cell density within the tumor parenchyma and reduced vascularization. Moreover, the expression of MCM7, an important subunit of MCM helicase, was significantly suppressed by cedrol in tumor tissue. Collectively, these results suggest that cedrol may act as a potential anti-cancer agent for colorectal cancer by inhibiting MCM protein expression and tumor growth.
Keywords: Apoptosis, Cedrol, Colorectal cancer, G1 arrest, Minichromosome maintenance proteins
Colorectal cancer is the third most common type of cancer worldwide, affecting all parts of the colorectal such as cecum, colon, rectum, anus and showing a high mortality rate due to difficulties in early detection and treatment [1,2]. Treatment strategies for colorectal cancer to improve the probability of survival include surgical treatments, such as resection, chemotherapy using cytotoxic drugs, and radiation therapies [3,4]. However, these treatment methods have been associated with various problems, mainly including side effects due to high toxicity, increased drug resistance, and metastasis . Thus, in recent years, there has been an increasing emphasis on developing natural substance-derived materials that yield fewer side effects, along with safe and excellent anti-cancer activity and various studies on the molecular mechanisms of compounds contained in natural products are actively underway . These natural compounds have various physiological activities such as anti-oxidant, anti-inflammatory, and anti-cancer effects and act selectively on cancer cells to inhibit proliferation and metastasis and to induce apoptosis. Apparently, many plant-derived drugs are known to exhibit anti-cancer activity by inhibiting cancer initiation, development, and progression . Among these phytochemicals, substances that are actually used as anti-cancer agents include vinca alkaloids, taxanes, epipodophyllotoxin, and camptothecins are used to treat various types of cancers, including breast, ovarian, and lung cancers, and Kaposi’s sarcoma [8-11].
Genomic instability is the hallmark of various cancers or cancer-related diseases with the increasing accumulation of DNA mutations or damage [12,13]. Thus, replication-related proteins or factors related to DNA repair can be targets for developing anti-cancer drugs . The minichromosome maintenance (MCM) protein is considered as an important first step of formation of pre-replication complex in eukaryotes, with six subunits (MCM2–MCM7) forming a heterohexameric complex [15,16]. Several MCM proteins are closely related to cancer formation, and its overexpression has been identified in many cancer tissues or cancer cell lines [17-22]. Among them, particularly MCM7 has a crucial role in tumor formation and progression based on its involvement in cellular proliferation by interacting with numerous proteins and a high expression in multiple malignancies . Therefore, the MCM protein is gaining attention as a biomarker for diagnosing cancers, as well as a target for cancer treatment . Our purpose is to explore substances that regulate the expression of MCM proteins for developing novel anti-cancer agents.
Cedrol used in this study was isolated from
HT29 cells were purchased from American Type Culture Collection (Manassas, VA, USA). HT29 cells were cultured in RPMI medium supplemented with 10% (v/v) FBS and 1% penicillin/streptomycin under standard culture conditions at 37°C and 5% CO2.
Cell viability was determined by the WST assay using the EZ-Cytox Cell Viability Assay Kit (Daeil Lab., Seoul, Korea). HT29 cells were treated with different concentrations of cedrol for 24 hours or 48 hours. The EZ-Cytox assay reagent (10 μL) was added to each cell culture well, and the mixture was incubated for 30 minutes at 37°C. The absorbance of each well was measured by a plate reader (Paradigm; Beckman Coulter, Brea, CA, USA) at the wavelength of 450 nm.
The effects of cedrol on the cell cycle progression were examined using the MUSE™ Cell Cycle kit (Luminex, Austin, TX, USA) according to the manufacturer’s instructions. Briefly, HT29 cells were treated with cedrol for 48 hours. The cells were then harvested and fixed in cold 70% ethanol for 3 hours at –20°C, followed by resuspending in PBS. After addition of an equal volume of Cell Cycle reagent, cells were incubated for 30 minutes at room temperature in the dark. Finally, flow cytometry was conducted (Muse™ Cell Analyzer; Luminex) at the Core Facility Center for Tissue Regeneration (Dong-eui University, Busan, Korea) and the Muse analysis software (version 1.9) (https://www.luminexcorp.com/muse-cell-analyzer/#documentation) was used to determine the relative DNA content.
Cell apoptosis was detected using an Annexin V & Dead Cell Kit (Merck Millipore, Darmstadt, Germany). Briefly, cells were treated with cedrol for 48 hours, harvested and then resuspended in PBS containing 1% FBS. Cells were stained with Annexin-V & Dead Cell reagent for 20 minutes under darkness at room temperature. The quantitative analysis of apoptosis was measured on a Muse™ Cell Analyzer (Merck Millipore).
HT29 cells were plated in 8-chamber slides and treated with various concentration of cedrol for 48 hours. The cells were fixed in 4% formaldehyde for 10 min at room temperature and then stained with DAPI (Sigma-Aldrich, St. Louis, MO, USA) for 10 min. After mounting, the stained cells were visualized under a fluorescence microscope (Carl Zeiss, Oberkochen, Germany) at the Core Facility Center for Tissue Regeneration (Dong-eui University).
Western blot analysis was performed according to our previous protocol . Cell lysates in Cytoskeletal buffer [10 mM Pipes (pH6.8), 100 mM NaCl, 1 mM MgCl2, 1 mM ethylene glycol tetraacetic acid, 1mM dithiothreitol, 1 mM phenylmethane-sulfonyl fluoride] containing 0.1% Triton X-100, 1 mM ATP and protease inhibitor were incubated on ice for 15 minutes, disrupted by sonication. For preparation of cytosolic proteins, the sonication process of the above was omitted. After centrifugation at 20,000
All animal experiments were performed under an experimental protocol approved by the Ethics Review Committee for Animal Experimentation of the Done-eui University (A2015-018). This study was conducted in accordance with the ARRIVE guidelines. Five-week-old athymic BALB/c-nu female mice (SLC, Hamamatsu, Japan) were maintained under specific pathogen-free conditions with a temperature of 22–24°C, humidity of 50%–60%, and lighting regimen of 12 hours light and 12 hours dark. HT29 cells (5 × 106 cells) were subcutaneously injected into the lateral flank of mice. When the tumor mass was palpable, 50 mg/kg of cedrol or 3 mg/kg of Adriamycin (Sigma-Aldrich) was administered intravenously three times a week for 14 days to the mice (n = 5 each group). All the mice were weighed, and the tumor volume was measured and then calculated using the formula: tumor volume (mm3) = [length × (width)2] × π/6. After 14 days of injection, the mice were sacrificed, and all tumors were removed and weighed.
To evaluate the histological changes, paraffin-embedded tumor blocks were sectioned at 5-µm thickness and stained with hematoxylin and eosin (H&E; Vector Laboratories Inc, CA, USA), followed by observation under a light microscope (Eclipse C; Nikon, Tokyo, Japan) at the Core Facility Center for Tissue Regeneration (Dong-eui University). For immunohistochemistry, sections were dewaxed, rehydrated, and boiled in antigen retrieving buffer (10 mM sodium citrate/0.05% Tween 20, pH 6.0) for 20 minutes. Sections were incubated with anti-MCM7 antibody (Santa Cruz Biotechnology) at 4°C overnight and further incubated with HRP-conjugated goat anti-mouse Immunoglobulin G (Santa Cruz Biotechnology) for 1 hour at room temperature, developed with diaminobenzidine substrate (Sigma-Aldrich). After counterstaining with hematoxylin, the sections were photographed under a light microscope (Eclipse C; Nikon).
The data were expressed as the mean ± standard deviation from at least three independent experiments. A student’s
To examine the inhibitory effect of cedrol on the HT29 cell proliferation, we performed a WST assay. Cells were treated with cedrol (Fig. 1A), incubated for 24 hours or 48 hours, and then the viable cells were measured. Compared to the control cells treated with vehicle, the cedrol-treated cell proliferation was significantly inhibited in a concentration- and time-dependent manner (Fig. 1B). When the concentration of cedrol was increased to 20, 25, and 30 μg/mL, the inhibitory rate of cell growth was increased to 33.64, 57.51 and 68.82%, respectively, after 48 hours of incubation.
Next, we investigated the effect of the cedrol on the expression of MCM proteins and PCNA, the proliferation markers, in HT29 cells based on our previous data (data not shown) that the methanol extract of
To demonstrate the molecular mechanism of suppression of HT29 cell proliferation by cedrol, we next examined the effects of cedrol on the cell cycle progression of HT29 cells. Figure 2A showed the increased distribution of cells in the G1 phases in a concentration dependent manner accompanied by a decrease in the S and G2/M phases, indicating that cedrol induces the G1 arrest. Compared to the untreated control, the treatment with 30 μg/mL of cedrol showed a significant increase in the G1 phase population from 56.36% to 73.26%, associated with the upregulation of p53 and p21, and downregulation of CDK2, cyclin E, pRb, and p-pRb (Fig. 2B and 2C).
Next, cedrol-mediated apoptosis was examined by flow cytometry using Annexin V/7-Aminoactinomycin D (7-AAD). As shown in Fig. 3A and 3B, the apoptosis of HT29 cells was induced by cedrol in a concentration-dependent manner. Live cells (Annexin V-/7AAD-) decreased from 89.65% to 65.49%, and apoptotic cells (Annexin V+) increased from 9.12% to 32.68%, after 48 hours incubation with 30 μg/mL of cedrol. Cedrol-mediated apoptosis was confirmed by observing the apoptotic bodies with chromatin condensation in a concentration-dependent manner using DAPI staining (Fig. 3C). Cedrol induced apoptosis of HT29 cells associated with cytosolic cytochrome-
To examine the antitumor effect of cedrol in a xenograft model tumor growth derived from HT29 cells was measured after treatment with cedrol or Adriamycin. As shown in Fig. 4A-4C, compared to vehicle-treated control, cedrol significantly inhibited tumor growth, showing the reduction of tumor volume and weight. Treatment with cedrol (50 mg/kg) reduced tumor volume more efficiently than Adriamycin (3 mg/kg) used as a positive control. Moreover, mice in the cedrol-treated group showed normal body weight whereas those in the Adriamycin-treated group exhibited body weight loss, suggesting that cedrol has an anti-cancer activity in vivo without any obvious toxicity (Fig. 4D).
To confirm the anti-cancer activity of cedrol in vivo, the histological and molecular changes were assessed in tumor tissue. After H&E staining, the cedrol-treated tumor showed remarkably reduced cell density both in parenchyma and stroma, and the blood vessels (arrows) in stroma were not easily detected compared with those in vehicle- or Adriamycin-treated tumor (Fig. 4E). Since MCM proteins were downregulated in HT29 cells by cedrol treatment, we next performed immunohistochemical staining with specific antibody against MCM7. As shown in Fig. 4E, although MCM7 expression was detected in vehicle- and Adriamycin-treated tumor tissue, the proportion of MCM7-positive cells was significantly decreased in cedrol-treated tumor tissue.
In the present study, anti-cancer efficacy of cedrol, an aromatic component of
MCM protein is a major replication protein involved in the early stages of DNA replication along with topoisomerase due to its helicase activity . It is attracting attention as a new target protein for anti-cancer drug development because of the recent characteristic overexpression in cancer cells. MCM proteins are essential for cell cycle progression, and they are overexpressed in various types of cancer cell lines . Cedrol exhibited a potent anti-proliferative effect through reduction of MCM proteins in HT29 colorectal cancer cells, resulting in cell cycle arrest and apoptosis. Cedrol-mediated G1 phase arrest is likely to be caused by a decrease in CDK2 and Cyclin E as a result of p53-dependent p21 upregulation. The Rb protein restricts the cell’s ability to replicate DNA, preventing the cell cycle progression from G1 to S phase . Phosphorylation of Rb, which is a signal downstream of the Cdk2/cyclin E complex, was inhibited by cedrol, which was associated with inhibition of MCM (MCM2–MCM7) protein expression . Cedrol-mediated apoptosis of HT29 cells occurred both internally by increasing cytoplasmic cytochrome
Moreover, we found that cedrol treatment suppressed tumor growth derived from HT29 cells and induced a number of histological changes in a xenograft tumor model. Compared to tumors in vehicle-treated mice, cedrol-treated mice showed a decreased tumor size and an indistinct tumor tissue structure as well as the decreased size and number of vessels in the tumor tissue. Angiogenesis plays an important role in tumor growth and metastasis in colorectal cancers; therefore, anti-angiogenesis is a promising approach for colorectal cancer therapy [33,34]. Our data on xenotransplantation are similar to previously reported data on the anti-angiogenic effect of widdrol, another active compound isolated from
In conclusion, we found that cedrol, an aromatic sesquiterpene compound present in
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (grant number: NRF-2017R1D1A1B04034994), Korea Basic Science Institute (National research Facilities and Equipment Center) grant funded by the Ministry of Education (2020R1A6C101A201) and Dong-eui University Grant (202201730001).
No potential conflicts of interest were disclosed.
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