記住我
ORIGINAL ARTICLE
Year : 2023 | Volume
: 9
| Issue : 4 | Page : 404-414
A preliminary study on anti-colorectal cancer effect and molecular mechanism of Aegiceras corniculatum extract
De-Chao Tan1, Xiao-Tao Hou2, Hua Luo3, Yi-Wei Chen2, Zheng-Cai Du2, Jin-Ling Xie2, Lin-Yao Wei2, Chi-Teng Vong4, Xiao-Yan Wen5, Er-Wei Hao2, Jia-Gang Deng2
1 Guangxi Key Laboratory of Efficacy Study on Chinese Materia Medica, Guangxi University of Chinese Medicine; Guangxi Collaborative Innovation Center of Research on Functional Ingredients of Agricultural Residues, Nanning; Institute of Chinese Medical Sciences, State Key Laboratory of Quality Research in Chinese Medicine, University of Macau, Macao; Sino-Canada Joint Zebrafish Lab for Chinese Herbal Drug Screening, Guangxi University of Chinese Medicine, Nanning, China
2 Guangxi Key Laboratory of Efficacy Study on Chinese Materia Medica, Guangxi University of Chinese Medicine; Guangxi Collaborative Innovation Center of Research on Functional Ingredients of Agricultural Residues; Sino-Canada Joint Zebrafish Lab for Chinese Herbal Drug Screening, Guangxi University of Chinese Medicine, Nanning, China
3 Guangxi Key Laboratory of Efficacy Study on Chinese Materia Medica, Guangxi University of Chinese Medicine; Guangxi Collaborative Innovation Center of Research on Functional Ingredients of Agricultural Residues, Nanning; Institute of Chinese Medical Sciences, State Key Laboratory of Quality Research in Chinese Medicine, University of Macau, Macao, China
4 Institute of Chinese Medical Sciences, State Key Laboratory of Quality Research in Chinese Medicine, University of Macau, Macao, China
5 Sino-Canada Joint Zebrafish Lab for Chinese Herbal Drug Screening, Guangxi University of Chinese Medicine, Nanning, China; Zebrafish Center for Advanced Drug Discovery, Keenan Research Center for Biomedical Science, Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada
Correspondence Address:
Dr. Er-Wei Hao
Guangxi Key Laboratory of Efficacy Study on Chinese Materia Medica, Guangxi University of Chinese Medicine, Nanning 530001; Guangxi Collaborative Innovation Center of Research on Functional Ingredients of Agricultural Residues; Sino-Canada Joint Zebrafish Lab for Chinese Herbal Drug Screening, Guangxi University of Chinese Medicine, Nanning 530200
China
Dr. Jia-Gang Deng
Guangxi Key Laboratory of Efficacy Study on Chinese Materia Medica, Guangxi University of Chinese Medicine, Nanning 530001; Guangxi Collaborative Innovation Center of Research on Functional Ingredients of Agricultural Residues; Sino-Canada Joint Zebrafish Lab for Chinese Herbal Drug Screening, Guangxi University of Chinese Medicine, Nanning 530200
China
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/2311-8571.391112
Objective: To study the inhibitory effects on colorectal cancer (CRC) and the underlying mechanism of the petroleum ether extract of Aegiceras corniculatum leaves (PACL). Materials and Methods: The effect of PACL on the proliferation of CRC cell lines DLD-1, HT-29, and SW480 was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay and colony-forming assay. And then, a wound-healing assay was used to measure the migration ability of three CRC cells. The cell cycle and apoptosis of three CRC cells were measured by PI/RNase staining and annexin V-FITC/double staining, respectively, and the intrinsic apoptosis pathway was studied by the Western blot. The anti-CRC effect of PACL in vivo was evaluated by HT-29 xenograft zebrafish embryos. Results: PACL inhibited cell viability and proliferation in DLD-1, HT-29, and SW480 cells in a dose- and time-dependent manner. PACL can inhibit cell migration in DLD-1 and SW480 cells but not in the less mobile phenotype cell HT-29. PACL treatment resulted in cell cycle arrest of DLD-1 and HT-29 cells in the G2/M phase. Moreover, PACL can induce apoptosis in all three CRC cells, which may be achieved by regulating the intrinsic apoptosis pathway mediated by mitochondria and the endoplasmic reticulum. Interestingly, the tumor sizes were decreased after treatment with PACL and PACL combined with fluorouracil in HT-29 xenograft zebrafish embryos. Conclusions: These findings suggested that PACL may exert its anti-CRC effect by inducing apoptosis through the intrinsic apoptosis pathway and show a significant anti-CRC effect in vitro and in vivo, so it might be potentially developed as an anti-CRC agent.
Keywords: Aegiceras corniculatum, apoptosis, cell cycle, colorectal cancer, zebrafish xenograft
Cancer is one of the diseases, with high morbidity and mortality worldwide. Globally, there were about 19.3 million new cancer cases, and 10.0 million cancer deaths occurred in 2020, and new cancer cases are projected to reach 28.4 million in 2040, a 47% increase from 2020.[1] In China, there were about 4.57 million new cancer cases and 3.00 million deaths recorded in 2020, according to the Global Cancer Observatory.[2] Globally, colorectal cancer (CRC) ranks second in mortality and ranks third in morbidity among all types of cancer for both sexes and all ages. Moreover, it is the second most common cancer in women and the third most common cancer in men.[1] CRC is prevalent in China; its estimated number of prevalent cases (5-year) was the highest of all types of cancer in 2020 for both sexes and all ages.[2] Epidemiological studies have shown that the incidence of CRC tends to occur in younger ages, which increased by 2% per year in individuals under the age of 50 in the recent 30 years.[3],[4] It is also happening in China that the incidence of CRC tends to occur at younger ages.[5] CRC is a solid malignant tumor, and 30%–50% of CRC patients still have local and systemic recurrence after receiving the preferred treatment method, surgical resection, and chemotherapy or radiotherapy. Moreover, local recurrence and metastasis are the leading causes of postoperative death.[4],[6] Therefore, better treatment for CRC is needed, and it is highly urgent to find effective and low toxicity of new anti-tumor drugs.
Mangrove plants grow in tropical and subtropical coastal intertidal zones and are an essential part of marine ecosystems. The natural environment of the intertidal zone is exceptionally harsh, with the characteristics of high salt, heat and humidity, and strong wind; this enables mangrove plants to protect themselves by synthesizing novel secondary metabolites.[7] Due to the extreme environment, mangrove plants are more likely to produce more bioactive components with novel structures than other terrestrial plants. There is a higher chance of discovering lead compounds for new drugs. In addition, mangrove plants are widely used in conventional medicine and can treat many ailments, including cancer, diabetes, leprosy, asthma, and hepatitis.[8] Based on the physiological specificity and traditional medical applications, mangrove plants have become attractive to scientists for in-depth research using modern medical research techniques. In recent years, studies have found that mangrove plants have a wide range of anti-tumor activities, and since then, many components with anti-tumor activities have been isolated.[9],[10],[11]
Aegiceras corniculatum (Linn.) Blanco (A. corniculatum), commonly known as black mangrove, river mangrove, or khalsi, is a mangrove shrub or small tree distributed widely in coastal and estuarine areas, distributed in India, Southeast Asia, Southern China, New Guinea, and Australia. This plant is recorded in many books as a medicinal plant, including “List of Guangxi Marine Medicinal Organisms,” “Chemistry for Marine Chinese Medicine,” and “Medicinal Plants in Australia,” and the medicinal parts are derived from the roots, bark, fruits, and leaves.[12],[13],[14] In “Chemistry for Marine Chinese Medicine,” it was written that A. corniculatum has the effects of analgesia, deworming, heat-clearing, and detoxification.[13] The traditional application of A. corniculatum is used to treat asthma, diabetes, rheumatism, and other diseases.[8] The leaf decoction or juice was utilized by Aboriginal Australians for the treatment of earache,[14] and the mashed fresh leaves were utilized by Jing people in China as a painkiller for sprain and strain.[15] Modern pharmacological studies have shown that A. corniculatum has anti-inflammatory,[16] analgesic,[17] anti-tumor,[18],[19] anti-bacterial,[20] anti-oxidative,[21] anti-diabetic,[22] and other pharmacological activities, so A. corniculatum has great medicinal value.
In the aspect of cancer, the extract of A. corniculatum has different degrees of inhibitory effects in different kinds of cancer cells in vitro, such as gastric cancer cells AGS, CRC cells HT-29, and breast cancer cells MCF-7 and MDA-MB-231.[23] Some chemical constituents with anti-tumor activity were isolated from A. corniculatum, including aegicoroside A, sakurasosaponin, sakurasosaponin methyl ester,[19] 2-hydroxy-5-ethoxy-3-nonyl1,4-benzoquinone, 5-O-butyl-embelin, 5-O-methylembelin, 5-O-methyl-rapanone, and 5-O-ethylembelin.[24] However, only a few systematic studies were focused on the anti-tumor effects of A. corniculatum and its active ingredients and their underlying mechanisms, so more studies are needed for a deep understanding of the anti-cancer effects of this plant.
Our previous studies demonstrated that the extract of A. corniculatum displayed varying degrees of anti-tumor activity in different kinds of cancer cells.[18],[25],[26] Among them, the petroleum ether extract of A. corniculatum leaves (PACL) inhibited cell proliferation in 18 cancer cell lines, and the half-maximal inhibitory concentration (IC50) values ranged from 28.82 to 301.36 μg/mL. These tumor cells are CRC cells HT-29, SW480, DLD-1 and COLO205, prostate cancer cells PC3 and DU145, ovarian cancer cells SKOV3-S and A2780, gastric cancer cells SGC-7901, tongue cancer cells Tca-8113, breast cancer cells MDA-MB-231 and MCF-7, liver cancer cells HepG2, SMMC-7721 and Bel-7402, cervical cancer cells Hela, bladder cancer cells EJ, and nonsmall cell lung cancer cells A549. However, no in-depth research was carried out in these studies. Therefore, in the present study, we investigated the effects of PACL on CRC cells and its potential mechanisms of action.
Materials and MethodsChemicals and reagents
RPMI 1640 medium was purchased from Wisent Bioproduct (Toronto, Canada). McCoy's 5A, L15 media, and phosphate-buffered salines (PBS) were purchased from KeyGEN BioTECH (Nanjing, China). Penicillin, streptomycin, fetal bovine serum (FBS), and 0.25% Trypsin-EDTA were obtained from Gibco Life Technologies (New York, the United States). Dimethyl sulphoxide (DMSO) was obtained from Solarbio life sciences (Beijing, China). 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) was obtained from Promega (Wisconsin, the United States). TGX Stain-Freei FastCast™ Acrylamide Kit was obtained from Bio-rad (California, the United States). The primary and secondary antibodies were purchased from Cell Signalling Technology (Massachusetts, the United States). Polyvinylidene fluoride (PVDF) membrane (Immobilon-P) was obtained from Millipore (Massachusetts, the United States). Fluorouracil (5-FU) was obtained from MedChemExpress (New Jersey, the United States). Vybrant™ Multicolor Cell-Labeling Kit was obtained from Thermo Fisher Scientific (Massachusetts, the United States). Ethyl 3-aminobenzoate methanesulfonate was purchased from Sigma (Missouri, the United States).
Cell culture
Three CRC cell lines, HT-29, SW480, and DLD-1, were purchased from Shanghai Cell Bank (Shanghai, China) and were incubated in McCoy's 5A, L15, and RPMI 1640, respectively. The other 15 cancer cells were also purchased from Shanghai Cell Bank. The cancer cells PC3, DU145, Bel-7402, EJ, SGC-7901, SMMC-7721, Tca-8113, and COLO205 were cultured in RPMI 1640, the cancer cells A2780 and MCF-7 were grown in DMEM, the cancer cells HepG2 and Hela were cultured in MEM, and the cancer cells MDA-MB-231, SKOV3-S, A549 were cultured in L15, McCoy'5A, and F12K, respectively. The medium contained 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% FBS, and the cells were maintained at 37°C and equilibrated with 5% CO2 and 95% air.
Animal study
According to the standard protocol, wild-type zebrafish lines were obtained from China Zebrafish Resource Center (Wuhan, China) and raised in a self-recirculating aquarium. The adult zebrafish were maintained at 28°C with a 14 h light and 10 h dark cycle and were fed with Hikari micro pellets and brine shrimp twice daily. The embryos were kept in embryo media at 28°C until they reached the desired embryonic stage. All animal procedures were carried out in compliance with the guidelines for scientific animal procedures approved by the ethics committee of the Guangxi University of Chinese Medicine, and the approval number is No. 2017-12-01-1.
Aegiceras corniculatum leaf extracts
The leaves of A. corniculatum were gathered from the Beilunhekou Mangrove Forest Reserve in Fangchenggang City, Guangxi, preserved at the Guangxi University of Chinese Medicine, and were processed to acquire PACL. The specific operation method and process were reported previously,[18] and all the extracts made were from the same batch.
Cell viability assay
HT-29, SW480, DLD-1, and NCM460 cells were tested for viability using an MTS kit, according to the kit's manufacturer's instructions. Cells were seeded in 96-well plates at a density of 3000 cells/well and allowed to attach overnight. Cells were then treated with the indicated doses of PACL for 24, 48 and 72 h. CellTiter 96® AQueous One Solution Reagent (WI, USA) was then added to the wells. After 2 h of incubation, measure the absorbance at 490 nm using a microplate reader. The Bliss method performed the calculation of IC50 in 18 cancer cell lines. The presentation of data was the mean of three independent experiments.
Colony-forming assay
Seeded the CRC cells in 6-well plates at 400 cells/well density. After 24 h, treat the cells with PACL (12.5, 25, 50 μg/mL), and use DMSO as a negative control. The indicated doses of PACL were changed after four days, and the experiment was terminated after the colony formation had been determined. The CRC cells were then fixed with 4% paraformaldehyde, and stained with crystal violet. A GelCount cell counter (Oxford, UK) was used to scan and capture the number of colony formations and then count the colony formation rate.
Wound healing assay
The CRC cells were placed in 6-well plates and cultured at 37°C until the cells became confluence. The wound was then created by scratching with a 200 μl pipette tip. Cells were washed with PBS to remove the cellular debris, and the live cells were treated with PACL. The cells were then photographed at 0, 12, 24, and 48 h after scratching. The wound closure rate was calculated, as explained previously.[27]
Cell cycle and apoptosis analysis
Seeded DLD-1, HT-29, and SW480 cells into the 25 cm2 flasks and treated with 25, 50, and 100 μg/mL of PACL or DMSO after culturing overnight. After the PACL treatment of 48 h, the cells were stained according to the instructions of the PI/RNase Staining Buffer kit (BD Biosciences, San Diego, the United States) or FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, San Diego, the United States). The data were acquired by Attune NxT flow cytometer (Thermo, Massachusetts, the United States) and analyzed by FlowJo software.
Western blotting
HT-29 cells were treated with PACL (12.5, 25, 50 μg/mL) or DMSO for 48 h before extracting the total protein. After lysing the cells, the protein was collected by a high-speed centrifuge at 4°C, and the protein concentration was determined by the BCA method. The protein samples were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, transferred onto PVDF membranes, blocked with nonfat milk at room temperature for 1 h, incubated with primary antibodies Caspase-3, Cleaved-caspase-3, Caspase-9, Cleaved-caspase-9, Caspase-12, total PARP, Bax, Bak and Bad, and then with secondary antibodies. The blots were developed by an ultra-sensitive enhanced chemiluminescence detection system.
Implantation of tumor cells into zebrafish embryos
The wild-type zebrafish embryos at 2-day post fertilization (dpf) were used for tumor cell injection. HT-29 cells were fluorescently labeled with DiI (Thermo, Massachusetts, the United States) according to the manufacturer's instructions. Approximately 400 labeled cells were injected into the yolk sac. At 4-hour post injection (hpi), 10 embryos with the same fluorescence intensity were selected for each group, and the embryos were divided into four groups: the control group, the PACL group (40 μg/mL), the positive control group (5-FU; 200 μg/mL) and the 5-FU + PACL group. The embryos were kept in an incubator at 34°C, with daily replacement of embryo water containing various factors, and on the third day of treatment (5-dpf) for photo observation. ImageJ was used to calculate the relative fluorescence intensity of cancer cells in the embryo and quantitatively evaluate the tumor growth.
Statistical analysis
All experiments were performed in triplicates unless stated otherwise. Statistical analysis was performed using GraphPad PRISM software version 7.00 (San Diego, California). The data were tested for significance using Student's t-test or one-way ANOVA. All quantitative data were expressed as mean ± standard deviation. Statistical significance was accepted when P < 0.05.
ResultsPetroleum ether extract of Aegiceras corniculatum leaves inhibited the cell viability of cancer cells
To study the effect of PACL on cell proliferation, treated the cells with indicated concentrations of PACL for 24, 48, and 72 h, and then the cell viability was tested by the MTS assay. As shown in [Table 1], PACL has varying inhibitory effects in 18 different cancer cell lines, and the IC50 values ranged from 28.82 to 301.36 μg/mL. Therefore, PACL exhibited varying degrees of anti-cancer activity in colorectal, prostate, ovarian, gastric, tongue, breast, liver, cervical, bladder, and lung cancer. This study was focused on the anti-CRC effect of PACL, so a normal human colon mucosal epithelial cell line, NCM460, was used as control cells.
Table 1: Half-maximal inhibitory concentration of inhibition of cell proliferation by petroleum ether extract of Aegiceras corniculatum leaves in cancer cells and normal cells NCM460 (µg/mL)As shown in [Table 1], PACL has significant anti-tumor activity against three CRC cells HT-29, SW480, and DLD-1. After the PACL treatment of 24, 48, and 72 h, the IC50 values of cell proliferation inhibition were 329.89, 118.33, 108.14 μg/mL in DLD-1 cells, whereas the IC50 values were 88.62, 49.54, 47.82 μg/mL in HT-29 cells, respectively. Besides, the IC50 values of cell proliferation inhibition were 163.10, 138.91, 125.23 μg/mL at 24, 48, and 72 h PACL treatment in SW480 cells, respectively. By comparing the IC50 values at 48 and 72 h among these 3 cell lines, PACL had the strongest inhibitory effect in HT-29 cells, followed by DLD-1 and SW480 cells.
As shown in [Figure 1]a, [Figure 1]b, [Figure 1]c, the cell viability was greatly decreased with increasing PACL concentration in DLD-1, HT-29, and SW480 cells, indicating that the inhibitory effects of PACL were concentration-dependent in these cells. By comparing the IC50 values in three treatment time points (24, 48, and 72 h), the inhibitory effects of PACL were enhanced along with more prolonged treatment time in DLD-1, HT-29, and SW480 cells [Table 1] and [Figure 1]a, [Figure 1]b, [Figure 1]c, indicating that the inhibitory effects were also time-dependent. On the other hand, the cell viability was also significantly decreased in a dose-and time-dependent manner in NCM460 cells [Figure 1d]. Taken together, it is suggested that PACL inhibited CRC cell proliferation in a dose-and time-dependent manner.
Figure 1: Petroleum ether extract of Aegiceras corniculatum leaves (PACL) reduced cell viability in colorectal cancer cells DLD-1, HT-29 and SW480, and normal cells NCM460. The cells were treated with 0-150 μg/mL of PACL for 24, 48 and 72 h. The cell viabilities of PACL were measured in (a) DLD-1 cells, (b) HT-29 cells, (c) SW480 cells, and (d) NCM460 cells. **P < 0.01, ***P < 0.001Petroleum ether extract of Aegiceras corniculatum leaves inhibited colony formation in colorectal cancer cells
To further validate the anti-proliferative activity of PACL in CRC cells, colony formation was also measured in HT-29, SW480, and DLD-1 cells. The cells were treated with 12.5, 25, and 50 μg/mL PACL. PACL reduced the number of colonies significantly in a dose-dependent manner [Figure 2]a, [Figure 2]b, [Figure 2]c, [Figure 2]d, [Figure 2]e, [Figure 2]f. The results showed that at 50 μg/mL, the colony numbers were at the lowest in these 3 cells. Besides, the colony number was significantly reduced at 12.5, 25, and 50 μg/mL PACL compared to control in DLD-1 and SW480 cells [Figure 2]a, [Figure 2]b, [Figure 2]e and [Figure 2]f. However, the colony number only decreased significantly at 25, 50 μg/mL PACL in HT-29 cells [Figure 2]c and [Figure 2]d. Therefore, it is further confirmed that PACL inhibited CRC cell proliferation in a dose-dependent manner.
Figure 2: Petroleum ether extract of Aegiceras corniculatum leaves (PACL) prevented colony formation in colorectal cancer cells DLD-1, HT-29 and SW480 cells. The cells were treated with 0-50 μg/mL of PACL. The clonogenic growth ability was measured in (a and b) DLD-1 cells, (c and d) HT-29 cells, and (e and f) SW480 cells. *P < 0.05, ***P < 0.001Petroleum ether extract of Aegiceras corniculatum leaves inhibited cell migration in colorectal cancer cells
The effect of PACL on the migration ability in CRC cells DLD-1, HT-29, and SW480 cells was studied by wound healing assay. As shown in [Figure 3]a, [Figure 3]b, [Figure 3]e and [Figure 3]f, 50 μg/mL PACL significantly inhibited the wound closure in DLD-1 and SW480 cells at 48 h, respectively, but not at 12 and 24 h, indicating it can inhibit the migration of DLD-1 and SW480 cells. However, 25 μg/mL PACL did not affect the percentages of wound healing in HT-29 cells at 12, 24, and 48 h [Figure 3]c and [Figure 3]d. There was no significant change in wound closure of HT-29 cells at 0 h and 48 h in the blank control group, suggesting that HT-29 cells don't have a very mobile phenotype. Taken together, it is suggested that PACL had a significant suppressive efficacy on the migration of mobile phenotype cancer cells DLD-1 and SW480.
Figure 3: Petroleum ether extract of Aegiceras corniculatum leaves (PACL) blocked cell migration in colorectal cancer cells DLD-1, HT-29, and SW480 cells. The cells were treated with 0-50 μg/mL of PACL for 12, 24 and 48 h. The percentages of wound healing were measured in (a and b) DLD-1 cells, (c and d) HT-29 cells, and (e and f) SW480 cells. **P < 0.01, ***P < 0.001Petroleum ether extract of Aegiceras corniculatum leaves affected the cell cycle distribution in colorectal cancer cells
After the PACL treatment of 48 h, measurement of the cell cycle distribution in CRC cells was performed by flow cytometry. As shown in [Figure 4]a, [Figure 4]b, [Figure 4]c, [Figure 4]d, as the concentration of PACL increased, the proportion of cells in the G0/G1 and G2/M phases decreased and increased in DLD-1 and HT-29 cells, respectively, while there was no significant change in the S phase. As shown in [Figure 4]a and [Figure 4]b, the cell populations in G0/G1 phase at 0, 25, 50 and 100 μg/mL were 71.11, 70.34, 66.76, and 54.36% in DLD-1 cells, respectively. And the cell populations in the G2/M phase at 0, 25, 50, and 100 μg/mL were 13.55, 13.06, 18.34, and 23.18% in DLD-1 cells, respectively. As shown in [Figure 4]c and [Figure 4]d, the cell populations in G0/G1 phase at 0, 25, 50, and 100 μg/mL were 79.80, 83.20, 75.10, and 58.31% in HT-29 cells, respectively. And the cell populations in the G2/M phase at 0, 25, 50 and 100 μg/mL were 8.73, 9.30, 14.92 and 16.94% in HT-29 cells, respectively. At 100 μg/mL PACL, the changes in G0/G1 and G2/M phases were significant in DLD-1 and HT-29 cells.
Figure 4: Petroleum ether extract of Aegiceras corniculatum leaves (PACL) interfered with the cell cycle in colorectal cancer cells DLD-1, HT-29, and SW480 cells. The cells were treated with 0-100 μg/mL of PACL for 48 h. The cell populations in G0/G1, S and G2/M phases were measured by flow cytometry in (a and b) DLD-1 cells, (c and d) HT-29 cells, and (e and f) SW480 cells. *P < 0.05, **P < 0.01. PACL: Petroleum ether extract of Aegiceras corniculatum leavesAs shown in [Figure 4]e and [Figure 4]f, the cell populations in G0/G1 phase at 0, 25, 50, and 100 μg/mL were 82.71, 74.31, 76.53, and 69.45% in SW480 cells, respectively, there was a slight decrease in the cell populations; however, the decrease was not significant. Similarly, PACL slightly increased the cell populations in S and G2/M phases, but the changes were insignificant. The cell populations were 6.21, 11.70, 11.02, and 15.07% in the S phase and 9.61, 12.08, 11.36, and 14.77% in the G2/M phase. Taken together, it is suggested that PACL arrested the cell cycle of CRC cell lines, DLD-1, and HT-29 cells in the G2/M phase.
Petroleum ether extract of Aegiceras corniculatum leaves induced apoptosis in colorectal cancer cells
As shown above, PACL significantly decreased cell viability at 48 h, so we next examined the effect of PACL on apoptosis in CRC cells DLD-1, HT-29, and SW480 cells. The cells were treated with PACL for 48 h, and apoptosis was detected and analyzed by flow cytometry. As shown in [Figure 5]a, [Figure 5]b, [Figure 5]c, [Figure 5]d, [Figure 5]e, [Figure 5]f, PACL could induce apoptosis in DLD-1, HT-19, and SW480 cells and its effect was enhanced with the increase in concentrations, indicating that it induced apoptosis in a dose-dependent manner. At 100 μg/mL, PACL significantly induced apoptosis in these 3 cells, but the proportion of apoptotic cells in SW480 cells was much lower than that in DLD-1 and HT-29 cells.
Figure 5: Petroleum ether extract of Aegiceras corniculatum leaves (PACL)-induced apoptosis in colorectal cancer cells DLD-1, HT-29, and SW480 cells. (a-f) The cells were treated with 0-100 μg/mL of PACL for 48 h. The percentages of apoptotic cells were measured by flow cytometry in (a and d) DLD-1 cells, (b and e) HT-29 cells, and (c and e) SW480 cells. (g) Representative immunoblots showed the expressions of apoptotic hallmarks. The cells were treated with 0–50 μg/mL of PACL for 48 h. *P < 0.05, **P < 0.01, ***P < 0.001Further investigation of the underlying mechanisms of PACL-induced apoptosis in CRC cells and the expressions of various apoptosis-related proteins were determined by western blotting in HT-29 cells. As shown in [Figure 5]g, treatment with PACL for 48 h significantly increased the expressions of Caspase-3, Cleaved-caspase-3, Caspase-9, Cleaved-caspase-9, Caspase-12, total PARP, Bax, Bak, and Bad in a dose-dependent manner. Therefore, PACL may induce apoptosis in CRC cell lines, DLD-1, HT-29, and SW480 cells.
Petroleum ether extract of Aegiceras corniculatum leaves reduced tumor growth and metastasis in zebrafish xenograft model
Further, we established a zebrafish xenograft model to evaluate the anti-CRC activity of PACL in vivo. HT-29 cells were labeled fluorescently with DiI and were injected into zebrafish embryos. [Figure 6]a and [Figure 6]b showed the picture of zebrafish with transplanted HT-29 cells on the day of injection or 3 days post-injection, respectively. In the control, PACL, and 5-FU groups, showed varying degrees of metastasis on the trunk [Figure 6]c. By comparing the fluorescence intensity from the control group, we found that the fluorescence intensities were decreased in PACL, 5-FU, and PACL + 5-FU treatment groups, indicating that all the treatment groups reduced tumor growth and metastasis in HT-29 xenograft zebrafish, and the combined treatment group had the most potent inhibition in cancer growth and metastasis [Figure 6]d.
Figure 6: Petroleum ether extract of Aegiceras corniculatum leaves (PACL) reduced tumor growth in HT29 xenograft zebrafish. (a) Image of HT-29 xenograft zebrafish embryo on the day of injection at 0 dpi (day postinjection). (b) Image of HT-29 xenograft zebrafish embryo at 3 dpi, the same embryo was used as in (a). (c) Representative images of HT-29 xenograft zebrafish embryos after 3 days of treatment in control, PACL, 5-FU (positive control) and PACL + 5-FU groups. (d) Relative fluorescence ratio of PACL and PACL + 5-FU treatment in HT-29 xenograft zebrafish embryos. *P < 0.05, **P < 0.01 Discussion Many preclinical studies and clinical studies have confirmed that traditional Chinese medicine plays an important role in anti-tumor, such as Xihuang Pills in the treatment of breast cancer and colon cancer,[28] Aidi Injection against liver cancer,[29] and Yiqi Qingdu Prescription in the clinical treatment of intermediate-stage and advanced non-small-cell lung cancer.[30] Marine Chinese medicine is an indispensable part of traditional Chinese medicine and plays a crucial role in the development of traditional Chinese medicine and because of the special growth environment, its anti-tumor activity is worthy of attention and study.
Marine Chinese medicine refers to the marine natural medicine used to prevent and treat diseases and health care problems under the guidance of the theory of traditional Chinese medicine. It is a rich library of medicinal resources, from which thousands of compounds with anti-tumor activity were identified, including psammaplin, didemnin B, dolastatin, lurbinectedin, plitidepsin, and halichondrin B.[31],[32] For example, cytarabine (Ara-C) is the first medicine isolated from marine Chinese medicine that has been modified to display significant anti-tumor activity and has been used clinically.[32] And trabectedin is isolated from sea squirt Ecteinascidia turbinate and has been used clinically to treat soft tissue sarcomas and other cancers under the trade name Yondelis.[33] In addition, many different compounds isolated from marine Chinese medicine (or structurally modified) have anti-tumor activities and have been in clinical trials, including disitamab vedotin, tisotumab vedotin, and depatuxizumab mafodotin.[32] However, some active substances isolated from marine Chinese medicines with anti-tumor activities have not yet entered clinical research. For example, the effect of gedunin isolated from Xylocarpus granatum on the cell cycle and apoptosis of ovarian cancer cells is related to the mitochondria-mediated apoptosis pathway.[34] Fucoidan extracted from brown seaweeds could inhibit the metastasis and invasion in MCF-7 cells by regulating the epithelial-mesenchymal transition signaling pathway.[35] Furthermore, A. corniculatum has been described in several pharmaceutical books as marine Chinese medicine,[12],[13] but its anti-tumor effect was not studied deeply. Therefore, the present study aimed to study the effects of PACL in CRC cells and its underlying mechanism.
The present study showed that PACL interfered with the growth and cell migration in CRC cells DLD-1, HT-29, and SW480 in vitro. It inhibited cell proliferation and migration in these three cells in a time-and concentration-dependent manner. This is consistent with previous studies demonstrating that A. corniculatum extract displayed potent anti-cancer activities in CRC.[18],[25]
In normal cells, the cell cycle regulates cell proliferation and DNA replication through a series of complex transduction signaling pathways, and this process also includes the repair of DNA damage, and cell death (apoptosis) will occur if DNA damage is not repaired. In tumor cells, there is abnormal regulation in the cell cycle due to gene mutation, resulting in excessive cell proliferation. The study of cell cycle regulation is a crucial anti-cancer research idea. Until now, some anti-cancer medicines have been developed for regulating the cell cycle, such as palbociclib, ribociclib, and abemaciclib.[36] In this study, our results showed that PACL could block the cell cycle in CRC cells DLD-1, HT-29, and SW480. It decreased the cell populations in G0/G1 phase, while it increased the proportion of cells in the G2/M phase in DLD-1 and HT-29 cells. In contrast, the n-butanol extract of A. corniculatum leaves (BACL) increased the cell populations in G0/G1 phase,[18] this difference in results was probably due to different types of extracts of A. corniculatum. Taken together, our current and previous studies suggested that A. corniculatum extracts could modulate the cell cycle. Therefore, future studies should focus on isolating the active components from these extracts to develop cell cycle-regulated anti-neoplastic agents.
The Caspase family is a group of proteases that exist in the cytoplasm and have similar structures, which are closely related to the apoptosis in eukaryotic cells and participate in the regulation of cell growth, differentiation, and apoptosis. Caspase-dependent apoptosis is one of the primary mechanisms to induce apoptosis, and it includes intrinsic and extrinsic pathways. The extrinsic pathway mainly refers to death receptor-mediated apoptosis, while the intrinsic pathway refers to mitochondrial-mediated apoptosis and endoplasmic reticulum-mediated apoptosis. In the present study, PACL was found to induce apoptosis in CRC cells DLD-1, HT-29, and SW480, and up-regulate the expressions of Caspase-3,-9, Cleaved-caspase-3,-9, and PARP in HT-29 cells. Interestingly, PACL could induce the activation of Bax, Bad, and Bak in CRC cells, suggesting that PACL induced apoptosis through the mitochondrial-mediated pathway, thus activating the intrinsic pathway. In mitochondrial-mediated apoptosis, the mitochondrial membrane potential decreases; this increases the mitochondrial membrane permeability and releases a large amount of cytochrome C. Cytochrome C and Caspase-9 form apoptotic bodies, which cleave and activate themselves, activating downstream Caspase-3 and triggering apoptosis.[37] In addition, the activation of Caspase-12 can also trigger apoptosis; it can further cleave and activate Caspase-3, thereby triggering apoptosis.[38] Our results showed that the expression of Caspase-12 was also up-regulated by PACL treatment in HT-29 cells, so we suggested that PACL could also induce apoptosis through the endoplasmic reticulum-mediated pathway. Similarly, it was shown that BACL could also induce apoptosis through the intrinsic pathway.[18]
Zebrafish have been an emerging animal model in the past two decades. It can be used in various anti-tumor studies, including tumor cell proliferation, invasion and introvation, metastasis, angiogenesis, and immune cell response.[39] In the present study, we further confirmed that PACL has anti-CRC activity in HT-29 xenograft zebrafish, and the combined treatment of PACL and 5-FU, a positive control, could enhance the effect of PACL. Similarly, our previous study demonstrated that NACL inhibited tumor growth in HT-29 xenograft mice.[18] This suggested that A. corniculatum extracts have potent anti-cancer activities.
In summary, the present study showed that PACL has significant anti-CRC activities in vitro and in vivo. It inhibited cell proliferation and migration in CRC cells DLD-1, HT-29, and SW480 cells in a time-and concentration-dependent manner. It also affected the cell cycle in G0/G1 and G2/M phases and induced apoptosis in a concentration-dependent manner. The induction of apoptosis was regulated by mitochondrial- and endoplasmic reticulum-mediated intrinsic apoptotic pathways. When used alone, PACL could inhibit tumor growth in HT-29 xenograft zebrafish, and when used in combination with 5-FU, 5-FU could enhance the anti-CRC effect of PACL. Further studies are warranted to isolate the active ingredients from PACL and investigate their effects and underlying mechanisms in cancer. Taken together, this study provides an important theoretical basis for the development of anti-CRC drugs from A. corniculatum.
Acknowledgments
We would like to acknowledge and thank Prof. Yi-Tao Wang for his wonderful help with revision.
Availability of data and materials
The datasets used in the current study are available from the corresponding author on reasonable request.
Financial support and sponsorship
This study was supported by grants from the Guangxi Science and Technology Plan Project (No. GKH15104001-11, No. GKAD19110155, No. 2018AD09008, 19-050-39), and the Guangxi Innovation Driven Development Project (No. GUIKEAA18242040).
Conflicts of interest
There are no conflicts of interest.
References
留言 (0)