Science
Alkaloid from Angelicae dahuricae Inhibits HeLa Cell Growth by Inducing Apoptosis and Increasing Caspase-3 Activity Kun Li,1,3 Qingwang Li,1,2 Zengsheng Han,1 Jian Li,1 Dawei Gao,1 Zhiwei Liu,1 Fulu Zheng4 (1Department of Biological Engineering, College of Environment & Chemical Engineering, Yanshan University, Qinhuangdao, The People’s Republic of China, 2College of Animal Science, Northwest A&F University, Yangling, The People’s Republic of China, 3College of Basic Medicine, Jiamusi University, Jiamusi, The People’s Republic of China, 4Renmin Hospital of Qinhuangdao, Qinhuangdao, The People’s Republic of China) DOI: 10.1309/XGVRQRG5GLKKYUQE
Abstract
Angelicae dahuricae has been extensively used in traditional Chinese medicine not only domestically for thousands of years but also in foreign countries such as Korea, Japan, and Russia. In this study, the effect of alkaloid from Angelicae dahuricae (AAD) on inhibiting HeLa
cell growth has been investigated by many approaches, including MTT, microstructure of apoptotic cells through DAPI staining and electron microscope, caspase-3 activity by chromotography, and cell cycle analysis by flow cytometry. The results showed that AAD could induce HeLa cell apoptosis, inhibit cell growth,
Cervical cancer is one of many malignant tumors threatening womens’ lives. In recent years, the death rate from cervical cancer has increased dramatically worldwide.1 The current cancer therapeutic approaches include surgery,2 chemotherapy, and radiotherapy; however, none of these cancer treatments can substantially cure the vertical of this cancer. Therefore, there’s a real demand to develop or discover an effective cancer therapeutic medicine and this is providing challenges to the life sciences. Although some drugs have been developed and may be effective to a certain degree,3,4 identifying new and highly effective medicines to improve the currently dismal survival rates for cervical cancer patients has a special significance. Angelicae dahuricae (AAD) is a valuable traditional Chinese medicine that is widespread in China. It has been used not only domestically for thousands of years, but also in foreign countries like Korea, Japan, and Russia. Pharmacologic study revealed that Angelicae dahuricae could treat the common cold, headache, nasal obstruction, rhinorrhea with turbid discharge, toothache, and fluor albus; could be used as a sunscreen with antiultraviolet properties; and to inhibit monophenolase.5 Therefore, many products have been developed, such as drug, health care, and cosmetology products.6 In recent years, researchers have found that Angelicae dahuricae could improve the human immune system, promote growth, enhance lung cell multiplication in hamsters and anti-tumor.7-8 Shangyuanqingxiang in Japan found that the methyl alcohol extraction of Angelicae dahuricae in fruit could be distributed in ADS-C and water layer, and when the water layer went through a Diaion pillar the ADS-C was acquired and could inhibit tumor cell growth. ADS-C was further purified using column chromatography and HPLC, and the ergosterol peroxide in these compounds had more restraining activity to 3 kinds of tumor cells: MK-1, B16F10, and HeLa cells. However, 3-hydroxide radical-P-methyl-1-alkene-6ketone had an effect on inhibiting B16F10 cell multiplication.9 The major active ingredients in Angelicae dahuricae are coumarin and naphtha, daucosterol, alkaloid, and necessary microelements in the body such as calcium, copper, iron, zinc, and nickel.10 In recent years, many scholars paid special attention to researching coumarin and naphtha in Angelicae dahuricae but there were no reports describing the effect of AAD’s natural properties as a cervical anticancer agent. 540
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and increase caspase-3 activity in a dose- and time-dependent manner. The mechanism of AAD-inducing apoptosis might be associated with multiple antiproliferative action towards the cells by inhibiting, preventing cell progression, and inducing a caspase-3 mediated apoptosis.
The purpose of this study was to investigate the effect of AAD on inhibiting cervical cancer growth and to provide strong scientific evidence for this application in the clinic. In the previous paper, we reported that procyanidins from Pinus koraiensis bark might have an cervical anticancer activity and the anti-hepatocellular carcinoma activity of alkaloid from oxytropis.11-12
Materials and Methods Chemicals The freeze-dried alkaloid flour from Angelicae dahuricae was prepared with Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco) stored at –20°C. Fetal bovine serum (FBS) was purchased from Hyclone, and DMSO, Trypsin, EDTA, MTT, and DAPI were all obtained from Sigma Chemical (St. Louis, U.S.A.). The caspase-3 kit used to detect apoptosis was purchased from Santa Cruz Biotechnology. All other chemicals were of analytical grade and supplied by Sigma Chemical. Preparation of Alkaloid Extracts Angelicae dahuricae was purchased from Minle drugstore of Qinhuangdao City of Hebei province, China. Authentication of plant material was carried out by Dr. Qingwang Li at the College of Animal Science, Northwest Agriculture and Forestry University, China, where the herbarium voucher is kept. Angelicae dahuricae (500 g) was minced and then soaked in water for 12 hrs. The aqueous phase was retrieved after the soaked sample was ultrasonically oscillated in distilled water (50 to 60) for 2 hours, followed by a second ultrasonic treatment. The water solution was then taken off and concentrated up to 1,750 mL by using a water heater. Concentrated sulfuric acid (96%) was added to the solution, and the pH value was adjusted to 2 to 3 pH. The supernatant was obtained and the residue removed, then the supernatant was centrifuged at 956 g for 10 min. Angelicae dahuricae was concentrated up to 400 mL by evaporation under reduced pressure and then extracted with concentrated sulfuric acid (96%) and n-butanol under ultrasound 25 times. The product was received and its pH labmedicine.com
Science value was adjusted to 9 to 10 with dense NaOH (50.5%), then the pH value was maintained between 2 and 3 by adding the n-butanol. The pH value of the extract was adjusted to 7 to 7.5, and the extract was concentrated by evaporation under a reduced pressure and put into a drying oven to cryodry. The crude alkaloid extract was obtained (10 g). Cell Culture HeLa cervical carcinoma cells were purchased from the Cell Center Institute of Basic Medical Sciences at XieHe Medical University. HeLa cell lines were cultured in RPMI-1640 medium supplement with 10% FBS, 100 U/μL of penicillin, and 100 U/μl of streptomycin at 37°C in a humidified incubator with 5% CO2. During the experiment, the medium was replaced by medium containing 50, 100, 200, and 400 μg/mL-1 AAD, which were dissolved in smaller quantities of ethanol (50%), then RPMI-1640 was added according to the concentration of AAD. Because Angelicae dahuricae was dissolved by ethanol (50%), the final ethanol concentration in each well of 6-well plates was 0.1% v/v. MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) Assay The capacity of AAD to interfere with the growth of HeLa cells was determined using the MTT dye assay. Cells were seeded in RPMI-1640 medium at a density of 5 × 104 cells/ mL in 96-well microplates for 12 h at 37°C, washed free with phosphate-buffered saline (PBS; pH 7.4), then the medium was removed and incubated with fresh medium with AAD (at final concentrations of 50, 100, 200, and 400 μg/mL-1) for an additional 24 h, respectively. Twenty μL of MTT was added to the medium and incubated for an additional 4 h, and the medium was removed. During this step, the MTT is converted to a blue formazan product by mitochondrial succinate dehydrogenase. This product was eluted from cells by adding 150 μL of DMSO, and absorbance at 540 nm was determined using an autoreader (model EL310 EIA). The concentration of AAD in each of the 8 replicate wells (1 row of a 96-well plate) was read and an average was determined. The rate of cell growth inhibition was calculated using the following formula: rate of cell growth inhibition = (C-T) / C × 100, where “C” and “T” mean average OD value of the control group and average OD value of the treated group. A regressive curve was determined for the rate of HeLa cell growth inhibition to drug-induced concentration. Analysis of Apoptotic Cells by DAPI Staining Cells (5 × 105 cells/well) cultured in the presence or absence of AAD for 48 hrs were collected, resuspended, and fixed in 4% paraformaldehyde in PBS. After PBS washing, cells were stained with DAPI for 30 mins, followed by another PBS washing. The stained cells were examined under a fluorescent microscope. Several independent experiments were subsequently performed for each concentration drug and time period (12, 24, and 72 hrs), and the apoptotic index (AI) was calculated: AI= the average apoptotic number of cells / the average total number of cells scored 100%.13 Ultramicrostructure of Apoptotic Cells by Electron Microscopy Cultured HeLa cells (5 × 106 cells/well) were collected in the control group (absence of AAD) and the treated group (400 μg/ mL-1) in a culture flask for 36 hrs at 37°C, centrifuged at 106 g for 5 min, supernatant was removed, and 3% glutaral was added. The cells were first fixed at 4°C for 1 night and once more with 1% osmic acid. Subsequently, the cells were embedded in Downloaded labmedicine.com from https://academic.oup.com/labmed/article-abstract/39/9/540/2504743 by guest on 07 August 2018
paraffin, sectioned (60 nm), and stained with uranyl acetate and lead citrate. The ultramicrostructure of the cells was examined under transmission electron microscopy (TEM). Caspase-3 Activity Assay Cultured HeLa Cells (5 × 105 cells/well) were collected in the control group (absence of AAD) and 3 treated groups (AAD: 100, 200, and 400 μg/mL-1) for 12, 24, 48, and 72 hrs. Caspase-3 activity was analyzed according to the manufacturer’s instructions in the caspase-3 colorimetric assay kit (R&D Systems). Cells were harvested, centrifuged at 125 g for 5 mins, lysed in 50 μL lysis buffer for 20 mins, vibrated for 10 s, and centrifuged at 10,621 g for 1 min at 4ºC. After centrifugation, the supernatant was collected and the protein concentration was determined. Each sample (50 μL) was incubated with caspase-3 substrate (5 μL) and 2× reaction buffer (50 μL) at 37ºC for 4 hrs and measured by chromotography at 405 nm wavelength. Flow Cytometry HeLa cells were exposed to the contractions of AAD (100, 200, and 400 μg/mL-1) for 12, 24, and 36 hrs, and the growth of the cells was analyzed using flow cytometry. During cell cycle, HeLa cells (1 × 106 mL-1) were exposed to 400 μg/mL-1 of AAD and fixed with 70% alcohol for 15 min at 4°C and incubated at –20°C for a minimum of 20 mins. The fixed cells were then washed twice, resuspended in PBS, and incubated at 37°C for 20 mins. The cells were stained with Annexin V-FITC (5 μL) and propidium iodide (50 μg/mL) for 15 mins and measured at 488 nm by flow cytometry within 6 hrs. The results were analyzed using Expo Software. Statistical Analysis Statistical analysis was performed using 1-way analysis of variance, and the differences between the means were tested using Duncan’s multiple range tests. Data was expressed as mean ± standard deviation (SD). P values of less than 0.05 were considered significant.
Results The Identification of Alkaloid The qualitative analysis of alkaloid was carried out with Mayer, Bertrand, and Sonnenschein precipitation reagent and revealed a light yellow, a brownish red, and a brownish yellow precipitation. The quality fraction mark of AAD was 99%.11 MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) Assay HeLa cells were treated with AAD of various concentrations (50, 100, 200, and 400 μg/mL-1) of AAD for 24 hrs, and cell viabilites were determined by MTT assay. As shown in Figure 1, AAD could inhibit the growth of HeLa cells in a dose-dependent manner. Cell growth was suppressed by AAD (50, 100, 200, 400 μg/mL-1) for 24 hrs. It was noted that AAD at 400 μg/mL-1 had an inhibitory effect of more than 40% on the HeLa cell growth in a 24-hr treatment (Figure 1). Analysis of Apoptotic Cells by DAPI Staining The HeLa cellular apoptosis was induced by AAD in a time- and dose-dependent manner. The AI of HeLa cells at 12 hours was 0.78 at the concentration of 100 μg/mL-1, 1.58 at September 2008 j Volume 39 Number 9 j LABMEDICINE
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Science Table 1_AI of HeLa Induced by AAD on Different Dosage at Different Times (% n=5) Different Control Time (h) Groups (µg/mL-1) 12 24
48
72
Control 100 200 400
1.92±0.01 2.76±0.02 4.28±0.03* 6.05±0.04*
2.66±0.22 3.79±0.04 5.78±0.05* 9.14±0.08*
0.09±0.01 0.78±0.02 1.58±0.01 3.24±0.04*
1.22±0.02 2.19±0.03 3.46±0.01* 5.76±0.03*
* P<0.01 as compared with control group.
Figure 1_Effect of AAD on HeLa Cell Growth Inhibition. HeLa cells are treated with AAD of various concentrations (50, 100, 200, and 400 μg/mL-1) for 24 h. Cell growth is suppressed by AAD (50, 100, 200, 400 μg/mL-1) for 24 h. It is noted that AAD at 400 μg/mL-1 has an inhibitory effect of more than 40% on the HeLa cell growth in 24-h treatment.
the concentration of 200 μg/mL-1, and 3.24 at the concentration of 400 μg/mL-1. The AI of different concentrations of AAD for 24, 48, and 72 hrs are represented in Table 1. Apoptosis increases with increasing concentrations and time-dependent treatment of AAD. The apoptotic features are present after 12 hrs, increased after 24 hrs and 48 hrs, and then significantly increased after 72 hrs. The morphological features of apoptosis were observed under fluorescence microscopy, including cell and nuclear shrinkage,
fluorescence intensity strength, nucleoli disappearance with crescent-like changes, and apoptosis-formed bodies. Necrotic cells indicated by PI staining were minor (between 4% and 6%) and there was no obvious relationship between the concentration and the reaction time of AAD (Table 1, Image 1). Ultramicrostructure of Apoptotic Cells by 221 Electron Microscopy After 36 hrs of HeLa cell exposure to AAD, the ultramicrostructure of HeLa cells using transmission electron microscopy started to show morphologic features of apoptosis, which manifested as the cell became round and shrunken in shape, subsequently losing contact with neighboring cells, with chromatin condensation and nuclear pyknosis, chondriosome scatter-like vacuolus, cell membrane blebbing, and irregular cellular shape, and finally floated into medium (Image 2).
Image 1_Apoptosis and necrotic HeLa cell (DAPI) (HE × 100 ). The morphological features of apoptosis were observed by fluorescence microscopy including cell and nuclear shrinkage, fluorescence intensity strength, disappearing nucleoli, crescent-like changes, and apoptosis-formed bodies. (A) Apoptosis of the HeLa cell (at 24 h) (arrow); (B) necrotic HeLa cell (at 24 h) (arrow); (C) apoptosis HeLa cell (at 48 h) (arrow); (D) necrotic HeLa cell (at 48 h) (arrow); (E) apoptosis HeLa cell (at 72 h) (arrow); and (F) necrotic HeLa cell (at 72 h) (arrow).
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Image 2_The morphological change of HeLa cell by AAD under electron microscope. After 36 hrs of exposure to AAD, the ultramicrostructure of the HeLa cells began to show morphologic features of apoptosis, which manifested as the cell became round and shrunken in shape, subsequently losing contact with neighboring cells, chromatin condensation and nuclear pyknosis, chondriosome scatter-like vacuolus, cell membrane blebbing and irregular cellular shape, and finally floated into medium by transmission electron microscopy. (A) A control cell (arrow) and (B) apoptotic cell (arrow).
Caspase-3 Activity Assay As shown in Table 2, the activities of 1 or more of caspase-3 were increased significantly in AAD-treated cells as compared with the activities in control cells. Caspase-3 activity was initially detected in a time-dependent manner, beginning to increase at 24 hrs and then peaking at 72 hrs. The dose-dependent manner in caspase-3 activities showed a 50% magnitude in cells treated at concentrations of 200 μg/mL-1. However, the activity of caspase-3 was shown to increase in the control cells following incubation for 48 hrs or longer (Table 2).
Table 2_Caspase-3 Activity of HeLa Induced by AAD on Different Dosages at Different Times Different Control Time (h) Groups (µg/mL-1) 12 24
48
72
Control 100 200 400
0.12±0.02 0.18±0.03 0.41±0.03* 0.51±0.05*
0.19±0.01 0.28±0.02 0.68±0.02* 0.89±0.09*
0.07±0.01 0.08±0.02 0.15±0.01 0.23±0.03*
0.09±0.02 0.12±0.01 0.23±0.02* 0.39±0.04*
* P<0.01 as compared with control group.
Flow Cytometry AAD could induce apoptosis of HeLa cells in a time- and dose-dependent manner. HeLa cells exposed to concentrations of AAD (100, 200, and 400 μg/mL-1) for 12, 24, and 36 hrs were analyzed using flow cytometry. HeLa cells became apoptotic at 12 hrs of exposure to AAD and peaked at 36 hrs. HeLa cells treated at 100 μg/mL-1 showed a small increase in apoptosis and then significantly increased with concentrations of AAD at 400 μg/mL-1 (Figure 2). The cell cycle analysis showed G2/M phase of 17.63% at 400 μg/mL-1 AAD for 12 hrs, and increased to 28.84% at 36 hrs; however, in the absence of AAD, the cell population was normal. Therefore, AAD might reduce reproductive activity of HeLa cells by inhibiting cell division (Table 3).
Discussion Apoptosis,14 or programmed cell death, is a highly regulated process that involves activation of a series of molecular events leading to cell death15-18 characterized by cellular morphological changes, chromatin condensation, and formation of apoptotic bodies associated with DNA cleavage into ladders.19-22 Apoptosis is responsible for the deletion of excess cells from normal tissue and for special pathologic events to occur.23 Although extracellular stimuli-induced apoptosis may involve Downloaded labmedicine.com from https://academic.oup.com/labmed/article-abstract/39/9/540/2504743 by guest on 07 August 2018
Figure 2_AAD Inducing HeLa Cell Apoptosis. HeLa cells are exposed to the concentrations of AAD (100, 200 and 400 μg/mL-1) for 12, 24, and 36 h. HeLa cells become apoptotic at 12 h of exposure to AAD and peaked at 36 h. The apoptosis shows an increase in HeLa cells treated at 100 μg/mL-1 and a significant increase in the concentrations of AAD (400 μg/mL-1).
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Science Table 3_The Change of HeLa Cell Ratio in Proliferation Cycle Induced by AAD (%) Phases
Control (24 h)
12 h
24 h
36 h
G0/G1 G2/M S G2/G1
59.26 9.86 15.88 1.66
51.44 17.63 14.67 1.67
47.36 18.37 18.98 1.71
44.26 28.84 11.67 1.66
multiple mechanisms,24 accumulated data suggests that mitochondria-initiated death pathway plays an important role in triggering apoptosis in response to these stimuli. In the mitochondriainitiated pathway,25-29 mitochondria undergoing permeability transition release apoptogenic protein such as cytochrome C or apoptosis-inducing factor (AIF) from the mitochondrial intermembrane space into the cytosol.30-31 Released cytochrome C can activate executioner caspase-3. Caspase-3 is an executioner caspase and exists in the cytoplasm as an inactive pro-caspase-3 that becomes proteolytically activated by multiple cleavages of its precursor 32 kDa to generate the 20/11 or 17/11 kDa active forms in apoptotic cells. After caspase-3 activation,32-35 some specific substrates for caspase-3 such as poly (ADP-ribose) polymerase (PARP) are cleaved and eventually lead to apoptosis.36-38 Evidence has shown that the possible mechanisms of various current antitumor drugs are related to their ability to induce apoptosis in target tumor cells.39-40 Therefore, induction of apoptosis has become a target strategy for antitumor drug discovery in recent years, and an apoptosis-inducing agent specific for tumor cells may be an ideal antitumor drug. Herbal therapy has thus been introduced partly because herbs consist of constituents with multiple targets, and partly because there is a long tradition of using herbs in Asia.41-42 The vast history of traditional Chinese herbs facilitates the identification of new compounds and novel action mechanisms in developing therapeutic drugs.43 We are interested in the bioactivities of alkaloid isolated from Angelicae dahuricae and have done a large scale screening test on its anticancer efficacies in human cervical cancer HeLa cells. Angelicae dahuricae is one of the commonly used herbal medicines of which the underlying mechanism is not clear. Recent investigations have shown that alkaloid isolated from Angelicae dahuricae could suppress tumor-promoting activity, induce apoptosis in HeLa cells, interfere with cell cycle progression, and suppress tumor growth. In this study, we tried to evaluate the ability of AAD to inhibit cell proliferation and induce apoptosis in HeLa cells and went further into some apoptosis-related events in these processes. This study has demonstrated that AAD was the most potent growth inhibitor against HeLa cells. Our results showed that AAD could induce apoptosis in HeLa cells revealed by the apoptotic alterations, including ultrastructural morphological changes by transmission electron microscopy. It inhibited the growth of HeLa cells in a dose- and time-dependent manner. Cell growth was suppressed by AAD (100, 200, 400 μg/mL-1) for 12, 24, 48, and 72 hrs. Apoptosis increased with rising concentrations of AAD and over time. We also detected a time- and dose-dependent increase in combined caspase-3 activity, beginning at 24 hrs of exposure to AAD and peaking at 72 hrs. The dose-dependent increase in caspase-3 activities showed a 50% increase in cells treated at 200 μg/mL-1. Therefore, AAD might inhibit HeLa cell growth 544
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Science by suppressing cell division and increasing the activities of caspase-3 to induce apoptosis. Taking all this data into account, we believe that AAD exerts multiple antiproliferative actions towards the cells by inhibiting and preventing cell progression, and inducing a caspase-3-mediated apoptosis. In conclusion, the results of this study provides scientific evidence to support the apoptosis-inducing activity of AAD and the involvement of caspase-3. More evidence demonstrating the effect on the apoptotic induction of AAD needs to be further evaluated. LM Acknowledgment: This study was supported by the Technology Committee of Qinhuangdao City. The authors would like to thank Prof. Zhangtao (Jiamusi Medical University, China) for providing the HeLa cell line in the present study.
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