Published online Feb 22, 2023.
https://doi.org/10.4163/jnh.2023.56.1.12
Antiproliferative effect of Citrus junos extracts on A549 human non-small-cell lung cancer cells
,1
and Young-Ran Heo2
Abstract
Purpose
This study investigates the alterations in A549 human non-small-cell lung cancer (NSCLC) cells exposed to Citrus junos extract (CJE). We further examine the antiproliferative and apoptotic effects of CJE on NSCLC cells.
Methods
Inhibition of proliferation was examined by applying the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) colorimetric assay on CJE-treated A549 NSCLC cells. The lactate dehydrogenase (LDH) assay was performed to measure the degree of toxicity of CJE on NSCLC cells. The effect on migratory proliferation was confirmed using the scratch wound healing assay. The antiproliferative effect of the CJE on human lung cancer cells was verified through morphological observation, fluorescence microscopy, and caspase-3 colorimetry.
Results
Exposure of NSCLC cells to CJE resulted in a dose- and time-dependent decrease in cell activity and increased toxicity to the cells. In addition, microscopic observation revealed a reduced ability of the cancer cells to migrate and proliferate after exposure to the CJE, with simultaneous morphological apoptotic changes. Fluorescence staining and microscopic examination revealed that this death was a process of self-programmed cell death of NSCLC cells. Compared to unexposed NSCLC cells, the expression of caspase-3 was significantly increased in cells exposed to CJE.
Conclusion
Exposure of A549 human NSCLC cells to CJE inhibits the proliferation, increases the cytotoxicity, and decreases the ability of cells to migrate and grow. Moreover, the expression of caspase-3 increases after CJE treatment, suggesting that the apoptosis of NSCLC cells is induced by a chain reaction initiated by caspase-3. These results indicate that Citrus junos is a potential therapeutic agent for human non-small-cell lung cancer.
INTRODUCTION
Citrus junos Sieb. ex Tanaka (Citrus ichangensis × Citrus reticulata var. austere) called “Yuja” in Korean, and “Yuzu” in Japanese, is a citrus species native to China [1, 2] and cultivated mainly in the southern coast of Korea and Japan. It is shaped like a yellow-golden round ball and has a unique sour taste and flavor. Citrus junos has traditionally been used as a medicinal material in Korea, Japan, and China [3]. Today, research on Citrus junos’s functional materials and their effects and applications have been extensively conducted. Studies on the health benefits of Citrus junos have been reported to have an inhibitory effect against several cancer cells [4, 5, 6, 7], hepatoprotective effects [8], antioxidant activity [4, 9, 10], and antidiabetic [7, 11, 12] and anti-obesity effects [13].
The main biologically active compounds contained in citrus fruits are vitamin C, E, dietary fiber, minerals, and flavonoids [10, 14]. According to related research, the major flavonoids and their quantities contained in an ethanol extract of Citrus junos are naringin (217 μg/mL), hesperidin (196.5 μg/mL), limonin (49.2 μg/mL), quercetin (42.9 μg/mL), catechin (7.2 μg/mL), nomirin (3.9 μg/mL), naringenin (3.2 μg/mL), and hesperetin (3.1 μg/mL) [7]. These flavonoids, well known as citrus fruit phytochemicals, are of great medical interest as they have a wide spectrum of biological activity, such as antiinflammatory, anticarcinogenic, neuroprotective, anti-allergic, estrogenic, antithrombotic, hepatoprotective, antibiotic, antiviral, antiulcer, antilipidemic and vasorelaxing properties [15]. Studies have especially reported that hesperidin [16], nobiletin [17], naringenin [18], quercetin [19], didymin [20], and kaempferol [21], which are the flavonoids of Citrus junos, have an inhibitory effect on lung cancer cells.
Lung cancer can be classified into two major histopathological subtypes: small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) [22]. Lung cancer is the leading cause of cancer death in the world [23], and NSCLC accounts for more than 80% of all lung cancer cases [24]. NSCLC is resistant to radiation and chemotherapy and is often discovered at too late of an advanced stage for surgical intervention [25]. Therefore, the overall survival rate of NSCLC at five years does not reach 15% [23, 25]. According to the data from the Korean Statistical Information Service (KOSIS, http://kosis.kr), lung cancer has hit the highest number of deaths (14,278–17,963 cases) among total cancer deaths (67,561–78,194 cases) in the past decade (2007–2016). The number of deaths due to lung cancer has been steadily increasing until recently.
The life and death of cells plays an important role in maintaining tissue homeostasis. Imbalance between cell proliferation and cell death results in serious disease, such as cancer [26, 27]. Cancer cells have a self-destruct mechanism called apoptosis, and caspases, a cysteine aspartyl-specific proteases play an important role in regulating cell death [28, 29, 30]. Cells initiate their own apoptotic death through the activation of these endogenous proteases, which can result in features such as cytoskeletal disruption, cell shrinkage, membrane blebbing, and nucleus condensation [26].
Inducing apoptosis in cancer cells is one of the most fundamental challenges in studying the anticancer effect [28]. Therefore, this study was conducted to verify whether Citrus junos extract (CJE) inhibits the proliferation of A549 NSCLC cells by inducing apoptosis.
METHODS
Preparation of CJEs
Citrus junos was purchased in Goheung, Korea. The seeds were removed, and the peel and pulp were freeze-dried (freeze dryer, Ilshin Biobase, Korea). The dried peel and pulp were pulverized and reflux condensate extracted in a 10-fold volume in 80% ethanol (1 L of 80% ethanol per 101.7 g of dried peel and pulp). The extraction was filtered (pore size 0.45 μm) and concentrated by evaporation (Rotary Evaporator N-1000, EYELA, Tokyo Rikakikai, Japan), and lyophilized again. The extraction yield was 47.3% on a dry weight basis (before extraction 101.7 g, after extraction 48.1 g). The CJE was stored at −80°C for further experiment.
Cell and culture conditions
The A549 cell line was obtained from the KCLB (Korean Cell Line Bank, Korean Cell Line Research Foundation, www.kclrf.org) and also provided by the biochemistry laboratory (School of Biological Sciences and Technology, Chonnam National University, Korea). Cells were used after thawing and passaged 1–3 times. Cells were maintained in an RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C with 5% CO2 in a humidified incubator (Jouan IGO150 CELLlife CO2 Incubator, Thermo Fisher Scientific, Waltham, MA, USA). All cell culture reagents were purchased from GIBCO Life Technologies (Thermo Fisher Scientific). The extracts were dissolved in dimethyl sulfoxide (DMSO). The cells were treated with 0.5% DMSO as the control group. The final concentration of DMSO in the culture medium was less than 0.5% (v/v).
Cell viability assay
Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) colorimetric assay. A Vybrant MTT Cell Proliferation Assay Kit (Thermo Fisher Scientific) was used to assess the effect of CJE on cell viability. Briefly, A549 cells were seeded at a density of 1 × 106/well in 96-well microplate and allowed to sit overnight. After the initial incubation period, cells were replaced with fresh culture medium and treated with increasing concentrations of CJE (0, 10, 20, 30, 40, and 50 μg/mL) for 12, 24, 48, and 72 hours. After each exposure time, cells were treated with 10 μL of MTT, 100 μL of SDS-HCl solution and incubated at 37°C for four hours. The cells were lysed in 100 μL of DMSO. The absorbance was measured at 570 nm on a microplate reader (Multiskan EX, Thermo Electron Corporation, Waltham, MA, USA).
Cytotoxicity assay
The Pierce LDH cytotoxicity assay kit (Thermo Fisher Scientific) was used to assess the toxic effect of CJE on A549 NSCLC cells, according to the manufacturer’s instructions. Lactate dehydrogenase (LDH) is a cytosolic enzyme used as an indicator to measure the degree of cytotoxicity. When the plasma membrane is damaged, LDH is released into the cell culture media. The released of LDH can be quantified by a coupled enzymatic reaction. First, LDH catalyzes the conversion of lactate to pyruvate via the reduction of NAD+ to NADH. Second, diaphorase uses NADH to reduce an iodonitrotetrazolium (INT) to a red formazan product. Therefore, the level of formazan formation is directly proportional to the amount of released LDH in the medium. To summarize the procedure of the LDH cytotoxicity assay, which was like to the MTT assay, the A549 cells were cultured in a 96-well microplate for one day and treated with CJE of a different concentration (0–50 μg/mL). We transferred 50 μL of the supernatant to a new 96-well microplate following exposure to the CJE for 24 hours. On that plate, 50 μL of the reaction mixture was added and incubated at room temperature for 30 minutes. After 50 μL of stop solution added, the absorbance was measured at 490 nm and 680 nm on the microplate reader. To determine the LDH activity, the absorbance value (background) at 680 nm was subtracted from the 490 nm absorbance before calculation of % cytotoxicity. To calculate the percentage of cytotoxicity, we subtracted the LDH activity of the spontaneous LDH release control (water-treated) from the sample LDH activity, divided it by the total LDH and multiplied it by 100:
Cell migration assay
Cell migration was evaluated using the scratch wound healing assay as previously described [17] with a slight modification. Cells were grown to 80% confluence in 6-well plate, and the wound was made by scratching the surface of the monolayer with a standard 200-mL pipette tip. The detached cells were removed by washing them twice with PBS, and the attached cells were incubated in a fresh medium with CJE (0–50 μg/mL). Following 24 and 48 hours of exposure, the cells were observed with a ×100 magnification using an inverted microscope (Eclipse TS100, Nikon, Tokyo, Japan).
Morphological analysis
We observed the morphological changes of the A549 NSCLC cells treated with CJE under an inverted microscope (Eclipse TS100, Nikon) and an IMT camera (Image and Microscope Technology camera, iCM3.0, Vancouver, BC, Canada). Following 24 and 48 hours of exposure, the cells were captured with a ×400 magnification using an IMT iCameras Version 4.0.
Fluorescence microscopy; Annexin V-FITC and PI
We used an annexin V-FITC apoptosis detection kit to observe the apoptosis of the A549 NSCLC cells. When the phosphatidylserine (PS) translocate from the inner face of the plasma membrane to the cell surface, it can be easily detected by staining it with a fluorescein isothiocyanate (FITC) conjugated annexin V. This protein has a high affinity for PS. The apoptosis and the necrosis of cells were visibly segregated by staining them with propidium iodide (PI), which binds to DNA in the nucleus. Cells were harvested after treated with 0–50 μg/mL concentration of CJE for 24 hours by centrifugation and resuspended them in 500 μL of binding buffer and added 5 μL of annexin V-FITC and 5 μL of PI. The cell suspension was placed on a glass coverslip and incubated for 15 minutes at room temperature in the dark. Cells were fixed with 2% formaldehyde before the coverslips were inverted into the glass slide for visualization. Cells were observed with a ×200 magnification using an inverted microscope (Leica DMIL LED, Leica, Wetzlar, Germany) and IMT i-Solution Auto Plus Version 22.1 was used to take pictures. The photograph was taken by setting the same exposure value (2,000 m/sec).
Expression level of caspase-3 analysis
To investigate the effect of the CJE on the programmed death of the A549 cells, the expression level of caspase-3 was measured. Caspase-3/CPP32 colorimetric assay kit was used. The A549 cells were cultured in 60-mm dishes and exposed to increasing concentrations of CJE for 24 hours (0–50 μg/mL). We collected the pellet of 1 × 106 cells and resuspended them in 50 μL of chilled lysis buffer, incubating the cells on ice for 10 minutes. The supernatant obtained by 1 minute of centrifugation (10,000×g) was transferred to a fresh tube and put on ice. The protein was then quantitated by the Bradford method [31] and diluted in 50 μL of cell lysis buffer per 50–200 μg of protein. The 50 μL of 2× reaction buffer (containing 10 mm of DTT), and 5 μL of DEVD-ρNA substrate were added and incubated at 37°C for 1 hour. The absorbance was measured at 405 nm using microplate reader.
Statistical analysis
All statistical analyses were conducted using SPSS 23 (IBM, Armonk, NY, USA). Differences due to CJE treatment were analyzed using one-way analysis of variance (ANOVA) and Scheffe’s post hoc test. Results were expressed as mean±standard deviation. We considered a p < 0.05 to be statistically significant.
RESULTS
CJE inhibits the proliferation of A549 NSCLC cells
As shown in Fig. 1A, CJE at 30, 40, and 50 μg/mL for 12 hours dose-dependently inhibited A549 cell growth by 99%, 79%, and 65% respectively as compared to the control cells. After 24 hours of exposure to CJE, the cell survival rate was significantly decreased (94%, 98%, 66%, 59%, and 57% of control cells) (Fig. 1B).
Fig. 1
CJE, Citrus junos extract. *p < 0.05, **p < 0.01, ***p < 0.001 as compared to the control.
Effects of CJE on cell viability in A549 NSCLC cells by different time points.
Cells were cultured in 96-well plates and incubated with solvent control (0.5% DMSO) or CJE 10–50 μg/mL for 12, 24, 48, and 72 hours (A-E). Following CJE treatment, MTT assays were performed, and the sample absorbance was measured at 570 nm on a microplate reader. Data was presented as mean ± SD (n = 3). The significance of the differences was analyzed by ANOVA.
Treatment with 20 μg/mL or more with CJE for 48 hours reduced the proliferation of lung cancer cells by up to 80%, 59%, 59%, and 57% as compared to control cells (Fig. 1C). Following 72 hours of exposure to greater than 20 μg/mL of CJE, viability of A549 was noticeably reduced (61–65% as compared to the control group) (Fig. 1D). The lowest survival rate was shown in a treatment of 50 μg/mL for 48 hours (57% as compared to the control) (Fig. 1C). Results showed that cell viability decreased as the concentration of CJE increased. We also observed a change in the lung cancer cell survival rate with an increase in treatment time (Fig. 1E). The evidence can be clearly seen in the 20 μg/mL treatment group. The survival rates of cells treated with 20 μg/mL of CJE were reduced to 92%, 73%, and 56% after 24, 48, and 72 hours, as compared to the 12-hour treatment group. Overall, the lung carcinoma cell survival rate was reduced depending upon the treatment time and the CJE dose. These results indicate that CJE inhibits the growth of A549 NSCLC cells.
CJE exerts cytotoxic effects on A549 NSCLC cells
LDH is a cytosolic enzyme that is released into the culture medium following the loss of membrane integrity that results from cell death [32]. We analyzed the CJE toxic effects on lung cancer cells by the quantification of LDH leakage from dead cells. The results of the cytotoxicity test were generally in contrast to those of the MTT cell survival test. The most prominent result was that as the concentration of the CJE was increased, the damage to the lung carcinoma cells was significantly increased. As shown in Fig. 2, the toxicity was 60, 100, 140, 180, and 160 times that of the groups treated with 10, 20, 30, 40, and 50 μg/mL of CJE, respectively, as compared to the control group (0.5% of DMSO). This result indicated that the treatment with CJE resulted in toxicity to carcinoma cells by inhibiting the integrity of the lung cancer cell membranes.
Fig. 2
CJE, Citrus junos extract.
Effect of CJE on cytotoxicity of A549 NSCLC cells.
The LDH cytotoxicity assay was conducted to evaluate the toxic effect of CJE on the A549 NSCLC cells. The most prominent result is that as the concentration of the CJE was increased, and the damage to the lung carcinoma cells was increased. The toxicity was respectively, 60, 100, 140, and 180 times higher in the 10, 20, 30, 40, and 50 μg/mL of CJE treatment groups as compared to the control group (0.5% of DMSO). The significance of the differences was analyzed using ANOVA and Scheffe’s post hoc test. Different alphabets indicate statistically significant differences (p < 0.05). Data was present as mean ± SD (n = 3).
CJE inhibits the migration of A549 NSCLC cells
We used the scratch wound healing assay to examine the effect of CJE on the movement of lung carcinoma cells. A scratch of a certain thickness was applied to the single-layer lung carcinoma cells and compared the restorative effect of scratches. As shown in Fig. 3, the scratch band became wider as the concentration of the CJE increased. The A549 NSCLC cells that had not been treated CJE or that had been treated with a low concentration of extract (10 μg/mL) recovered from their wounds over time and moved closer to each other. As the exposure time increased, the width of the scratch on the A549 NSCLC cells exposed to 20 μg/mL of CJE became wider, and the density of the attached cells was markedly lowered. In the group that had been treated with 20 μg/mL for 48 hours, the wound thickness was less than in the group treated with 20 μg/mL for 24 hours. However, the interval between each cell was remarkably widened. It can be considered that there was a slight cell migration, but the number of cells did not increase.
Fig. 3
CJE, Citrus junos extract.
Effect of CJE on cell migration of A549 NSCLC cells.
Cell migration was evaluated using a scratch wound healing assay. Cells were grown to 80% confluence in a 6-well plate, and a wound was made by scratching the surface of the monolayer with a standard 200 μL pipette tip. The detached cells were removed by washing them twice with PBS, and the attached cells were incubated in a fresh medium of CJE (0–40 μg/mL). Photographs were taken after 24 and 48 hours by inverted microscope (×100).
CJE changes the morphology of A549 NSCLC cells
Observation of the morphological changes in cells is the most basic and important step in cell experiments. The morphological cell changes by CJE treatment were analyzed. The shape and cohesion of the cells and the degree of attachment to the dish were changed. As in the MTT and wound recovery trial, those cells with 10 μg/mL extract treatment were not inhibited by the CJE treatment. On the other hand, after treatment with 20 μg/mL for 48 hours, the number of cells attached to the plate was significantly reduced. Morphological changes in the cells treated with a concentration of 30–40 μg/mL of extract can be identified, not only by the density, but also by the cell shape after 24 hours. A characteristic morphological change of cell death, small swelling, was observed. As shown in Fig. 4, going down to the right, the pictures show that the cell shape changes from a polygon forming a lump to a primitive circular shape. Cell membrane blebbing was also detected.
Fig. 4
CJE, Citrus junos extract.
Effect of CJE on cell morphology of A549 NSCLC cells.
The shape and cohesion of the cells and the degree of attachment to the dish were changed. The cells with 10 μg/mL extract treatment were not inhibited by the CJE treatment. On the other hand, after 48 hours following the 20 μg/mL treatment, the number of cells attached to the plate was significantly reduced. Morphological changes of the cells treated with a concentration of 30–40 μg/mL extract can be identified, not only by density but also by the shape of cells after 24 hours. Photographs were taken at 24 and 48 hours by inverted microscope (×400).
CJE induces apoptosis in A549 NSCLC cells
We used fluorescence microscopy to investigate the effect of CJE on apoptosis or necrosis of A549 NSCLC cells. Fig. 5 shows the lung cancer cells treated with (A) 0.5% DMSO (control), (B) 10 μg/mL of CJE, (C) 20 μg/mL of CJE, and (D) 30 μg/mL of CJE that had been stained with annexin V-FITC and PI. In the 10–30 μg/mL treatment group, a green light was expressed more than in the control group, and red light was not expressed. The brightest green light was expressed in the cells treated with 20 μg/mL of CJE. These results suggest that apoptosis was induced in A549 NSCLC cells by the effect of treatment with CJE.
Fig. 5
CJE, Citrus junos extract.
Induction of apoptosis by CJE on A549 NSCLC cells.
A549 lung cancer cells were grown and treated with (A) 0.5% DMSO (control), (B) 10 μg/mL, (C) 20 μg/mL, (D) 30 μg/mL of CJE. In the 10–30 μg/mL treatment group, green light was more pronounced than in the control group, and red light was not expressed. Photographs were taken after 24 hours by an inverted microscope with an LED lamp (×200). Green light (apoptotic cell); annexin V-FITC, red light (necrotic cell); PI.
CJE activates the caspase-3 on A549 NSCLC cells
The mean absorbances measured at 405 nm following 24 hours treatment with 0 (control), 20, 40, and 60 μg/mL of CJE were 0.148, 0.172, 0.168, and 0.165, respectively. Fig. 6 shows the difference in the ratio to the control. The ratio of caspase-3 activation in the control group was 116%, 113%, and 111%, in order of increasing concentration of CJE. The highest activity of caspase-3 was observed in the group treated with 20 μg/mL of CJE and showed a significant difference. The data indicates that CJE-induced apoptosis in A549 NSCLC cells is associated with the activation of caspase-3.
Fig. 6
CJE, Citrus junos extract.
Effects of CJE on the expression level of caspase-3 in A549 NSCLC cells.
The ratio of caspase-3 activation to the control group was 116%, 113%, and 111% control, in the order of increasing concentration of CJE. The highest activity of caspase-3 was observed in the group treated with 20 μg/mL of CJE and showed a significant difference. The differences were analyzed using ANOVA and Scheffe’s post hoc test. Different alphabets indicate statistically significant differences (p < 0.05). Data was presented as mean ± SD (n = 3).
DISCUSSION
Through a cell viability and cytotoxicity assay, we confirmed that the effect of inhibiting proliferation of lung cancer cells by CJE treatment was concentration and time dependent. These findings agree with previous reports on the antiproliferative effects of CJEs at a range of concentrations (2.5–25 μg/mL) in human prostate cancer cells [4]. It is known that the components contained in Citrus junos, such as hesperidin [16], nobiletin [17], naringenin [18], quercetin [19], didymin [20], and kaempferol [21] have inhibitory effects of NSCLC cells. These substances are revealed to inhibit proliferation by inducing apoptosis in lung cancer cells, and the mechanisms are as follows. Hesperidin increased caspase-3 expression and decreased mitochondrial membrane potential [16], and naringenin increased DR5 (death receptor 5) expression and enhanced TRAIL (tumor necrosis factor related apoptosis inducing ligand)-induced apoptosis [18]. Quercetin suppressed the proliferation of lung cancer cells by reducing the activity of aurora B kinase [19], and didymin induced apoptosis by activating the Fas/Fas ligand system [20]. Kaempferol and nobiletin inhibits MAPK (migogen-activated protein kinase) to induce apoptosis and suppress proliferation of lung cancer cells [17, 21]. Although only caspase-3-induced apoptosis was confirmed in this study, CJE could inhibit the growth of A549 cells the complex action of these substances.
The cytotoxicity test showed an opposite effect from the cell activity test, and the toxicity to the lung cancer cells increased as the concentration and exposure time of CJE increased. This indicated that the treatment with CJE resulted in toxicity to carcinoma cells by inhibiting the integrity of the lung cancer cell membranes. These results are similar to the studies of Citrus aurantiifolia (English name: lime) extracts on human colon cancer cells, which showed higher LDH releases as compared to the positive control [33]. These studies demonstrate that citrus-derived materials are useful in inducing cytotoxicity and reducing the viability of cancer cells by damaging cell membrane integrity.
A wound recovery test verified that the ability of lung cancer cells to migrate and proliferate was reduced by the treatment with CJE. This result is similar to the findings of the previous study on the anti-metastatic activity of 20–100 µm of nobiletin in human glioma cells [17]. Furthermore, the cells exposed to a CJE concentration of 30 μg/mL or greater at 48 hours were no longer able to be observed by 100× microscope objective. This indicates that CJE may have an anti-migration effect on NSCLC cells.
We confirmed that the density of A549 cells decreased through cell morphological changes by treatment with CJE. This result is similar to the findings of the previous study in which the reduction of lung cancer cells and apoptotic morphologic changes were observed after treatment with kaempferol [21].
Annexin V is a protein present in the intracellular membrane possessing a high affinity for PS. It emits a bright green light from the labelled FITC by attaching to PS at the early apoptotic period when the membrane is still intact. PI penetrates the collapsed membrane of the dead (necrotic) cells and stains the DNA in the nucleus to emit a red light [32]. The cells treated with over 40 μg/mL of CJE were not fixed on the glass coverslip and were almost lost. So, data could not be displayed.
Caspase activation is a major feature of apoptosis [32]. The caspase-3 is an apoptosis executioner [27, 28, 29], which results in a proteolytic cascade that arise to a cell death phenotype. It is featured by DNA fragmentation, chromatin condensation, cell shrinkage and membrane blebbing [30]. As shown in Fig. 1, cell proliferation was inhibited by treatment of CJE, and morphological characteristics of cell death were observed in Fig. 4. And the apoptosis signal was confirmed by a green light in Fig. 5. Caspase-3 colorimetry was conducted to determine whether these changes were caused by the programmed death of A549 NSCLC cells associated with caspase-3 after treatment with CJE.
SUMMARY
This study was conducted to investigate the inhibitory effect of CJE on the proliferation of A549 human NSCLC cells. As the concentration of CJE and the exposure time increased, lung cancer cell proliferation decreased, and the cytotoxicity level increased. After treatment with CJE, A549 cells' morphological change and the decrease of growth range and amount were confirmed. In addition, one of our hypotheses that self-planned death of A549 NSCLC cells was induced by CJE treatment was proved through the results from fluorescence staining and caspase-3 expression. Taken together, CJEs may be useful therapeutic agent for non-small-cell lung cancer.
Conflict of Interest:There are no financial or other issues that might lead to conflict of interest.
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