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Litchi seed extract inhibits epidermal growth factor receptor signaling and growth of Two Non-small cell lung carcinoma cells
BMC Complementary and Alternative Medicine volume 17, Article number: 16 (2017)
Litchi seeds possess rich amounts of phenolics and have been shown to inhibit proliferation of several types of cancer cells. However, the suppression of EGFR signaling in non-small cell lung cancer (NSCLC) by litchi seed extract (LCSE) has not been fully understood.
In this study, the effects of LCSE on EGFR signaling, cell proliferation, the cell cycle and apoptosis in A549 adenocarcinoma cells and NCI- H661 large-cell carcinoma cells were examined.
The results demonstrated that LCSE potently reduced the number of cancer cells and induced growth inhibition, cell-cycle arrest in the G1 or G2/M phase, and apoptotic death in the cellular experiment. Only low cytotoxicity effect was noted in normal lung MRC-5 cells. LCSE also suppressed cyclins and Bcl-2 and elevated Kip1/p27, Bax and caspase 8, 9 and 3 activities, which are closely associated with the downregulation of EGFR and its downstream Akt and Erk-1/-2 signaling.
The results implied that LCSE suppressed EGFR signaling and inhibited NSCLC cell growth. This study provided in vitro evidence that LCSE could serve as a potential agent for the adjuvant treatment of NSCLC.
Lung cancer is the most threatening type of cancer in industrial countries, with high morbidity and mortality, because many patients are diagnosed with advanced disease . Non-small cell lung cancer (NSCLC) is the most prevalent type of lung malignancy. Although recent advancements in treatment strategies using tyrosine kinase inhibitors (TKIs) to treat NSCLC have been developed to prolong patient survival by 3-fold as compared to traditional chemotherapies, drug resistance still occurs in a proportion of patients. Other adjuvant strategies to prolong the survival rate of NSCLC patients administered TKIs should be developed [2–4]. Litchi (Litchi chinensis, Sapindaceae) is a warm-climate fruit tree that originates from southern China and is cultivated in semi-tropical areas of the world for its delicious fruit . In traditional Chinese herbal medicine, the function of litchi seeds are the release of stagnant humor and remote chilling, and they are used as analgesic agent capable of relieving cough, gastralgia, neuralgia, orchitis, colic, testicular swelling and smallpox . In India, the seeds are powdered and administered to people with intestinal trouble. The seeds also have the reputation of relieving neuralgic pain in China. In recent studies, a variety of proanthocyanidins, flavonoid glycosides and polysaccharides were identified from litchi seeds [7–9]. Some of these compounds appeared to exhibit anti-neoplastic activities in breast cancer, sarcoma, nasopharynx cancer, lung cancer, cervical cancer and hepatocellular carcinoma cell lines [7, 8, 10, 11]. A possible mechanism may be due to the inhibition of EGFR signaling and concomitant induction of intrinsic and extrinsic apoptosis pathways . The hypothesis of the present study is the potential application of litchi seed extract (LCSE) as an anti-lung cancer agent because of the ability to target EGFR. However, the detailed cellular and molecular mechanisms of the suppressive effect of LCSE in NSCLC cells are still unclear. In this study, we investigated the effects of LCSE on two NSCLC cell lines, adenocarcinoma A549 and large-cell carcinoma NCI-H661 cells, to evaluate the potential of LCSE for the treatment of NSCLC.
Chemicals used for the preparation of buffers, cell-staining fluorescent dyes or drugs such as proteinase inhibitor cocktail, sodium orthovanadate, sodium fluoride, sodium pyrophosphate, Triton X-100, ammonia persulfate, rhodamine 123, propidium iodide, N,N,N’,N’-tetramethylethylenediamine, sodium dodecyl sulfate (SDS), Tween 20, gallic acid and catechin were obtained from Sigma (St. Louis, MO, USA). Cell-culture materials such as Roswell Park Memorial Institute (RPMI) media 1640, Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), L-glutamine, trypsin and antibiotics were purchased from Gibco Ltd. (Paisley, UK). The protein assay kit (bicinchoninic acid; BCA) was purchased from Pierce (Rockford, IL, USA). Acrylamide was obtained from Bio-Rad (Hercules, CA, USA). Polyvinylidene fluoride (PVDF) membrane (Immobilon-P) was obtained from Millipore (Bedford, MA, USA). Mouse monoclonal anti-caspase 3, B cell lymphoma 2 (Bcl-2), cyclin B1, cyclin D1, cyclin E and anti-MAP kinase (Erk-1/-2) antibodies and rabbit anti-phosphor-Erk-1/-2 (The202/Tyr204) were purchased from Zymed (San Francisco, CA, USA). Rabbit polyclonal anti-human pan Akt, anti-phosphor-Akt (S473), anti-EGFR, anti-phosphor-EGFR (phospho Tyr1092) and anti-Kip1/p27, and goat polyclonal anti-pro-caspase 8, pro-caspase 9, poly [ADP-ribose] polymerase (PARP), Bcl-2 associated protein X (Bax) antibodies and goat anti-rabbit, anti-mouse and rabbit anti-goat secondary antibodies conjugated with horseradish peroxidase (HRP) were obtained from R&D Systems (Minneapolis, MN, USA). Annexin V conjugated with fluorescein isothiocyanate (FITC) was obtained from Gene Research (Taipei, Taiwan).
Litchi seed extract
Hei Yeh Litchi (Litchi chinensis Sonn. var. Hei Yeh) fruit were purchased from Rayfoung Co., Ltd (Chiayi, Taiwn) and identified by Dr. Chih-Cheng Lin and Chih-Ping Hsu using the Digital Fruit Genetic of Taiwan database of the Agricultural Research Institute (Council of Agriculture, Executive Yuan of Taiwan) as a reference (https://kmweb.coa.gov.tw/subject/ct.asp?xItem=176011&ctNode=5525&mp=1&kpi=0&hashid=). Litchi seed extract was obtained using the method described in a previous report . Briefly, litchi seeds dried in a 70 °C oven were ground using a stainless-steel grinder (RT-02, Rong Tsong Iron Factory Incorporation, Taiwan). Crude extract of litchi seeds was obtained by mixing the powder with 70% ethanol and refluxing overnight. The solution was then filtered and centrifuged to remove any undissolved materials. The supernatant was subsequently concentrated until no ethanol remained using a rotary evaporator under reduced pressure and a water bath <35 °C, which was then freeze-dried. The final crude extract was defined as LCSE. The total levels of phenols, flavonoids and condensed tannins were estimated using colorimetric methods as described previously .
A549 and NCI-H661 cells were purchased from the Bioresource Collection and Research Center in Taiwan. A549 cells were established from lung carcinomatous tissue from a 58-year-old Caucasian male, and the cell type was identified as lung carcinoma. NCI-H661 cells were derived from the lymph node of a patient with large-cell lung cancer. These two cell lines were cultured in 90% RPMI 1640 with 2 g/L sodium bicarbonate, 10% heat-inactivated FBS, 25 U/mL penicillin and 25 μg/mL streptomycin. The cells were incubated at 37 °C in a 95% air/5% CO2 water-saturated atmosphere. All experiments were carried out using cell lines passaged between 5 and 20 times.
Cell proliferation assay
Cells were plated at 100,000 cells per 60-mm tissue culture dish and then treated with LCSE (0, 12.5, 25, 50, 100, or 150 μg/mL) after approximately 18 h, when the cells had become attached to the bottom of the plates. Cells were incubated with LCSE for 24 h and then collected by trypsinization, stained with trypan blue, and counted in suspension in duplicate using a hemocytometer. Data were obtained from the averages of three independent experiments.
Clonogenic growth assay
200 cells were seeded in a 6-well plate and treated with LCSE (1 ~ 50 μg/mL) then incubated at 37 °C for 14 days. On day 14, the colonies were fixed in 70% ethanol and stained with 0.2% crystal violet. Colonies of >50 cells were counted and the colony-forming potential of LCSE-treated NSCLC cells was expressed as a percentage of colonies of the untreated cells.
As described in a previous report , LCSE-treated cells were collected by trypsinization and then fixed in 70% ethanol at −20 °C for at least 30 min. Fixed cells were reconstituted in phosphate-buffered saline and then stained with propidium iodide solution (20 μg/mL propidium iodide and 10 μg/mL RNase A) at 37 °C in the dark for 30 min. The cell cycle of LCSE-treated cells was examined by flow cytometry (Becton Dickinson, CA) using FL-2A to score the DNA content of cells. The numbers of cells in the G1, S and G2/M cell-cycle phases were determined using Modfit software and expressed as the percentage of total cells (Verity Software House, Inc., Topsham, ME, USA).
Apoptosis of LCSE-treated cells was analyzed using annexin V-FITC labeling followed by flow cytometry, as described in previous reports . The treated cells were trypsinized and suspended in binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2). Cells were stained with 2 μg/mL annexin V-FITC at room temperature in the dark for 30 min. The fluorescence intensity of the labeled cells was measured using FITC and flow cytometry with FL-1H. Untreated cells served as the negative control.
Mitochondrial membrane potential (Δψm)
Δψm, the mitochondrial membrane potential, indicates the mitochondria membrane integrity in cells, and was assessed as described by Hsu et al. . Briefly, LCSE-treated cells were collected, suspended at a density of 1 × 106 cells/mL in fresh medium and stained with 10 μg/mL rhodamine 123 for 30 min at 37 °C. Cells were then washed twice with fresh medium, and the fluorescence intensity of the cells was immediately examined by flow cytometry using FL-1H. Ten-thousand cells without cell debris were analyzed, and the rhodamine 123-negative cells were defined as those with a lower fluorescence intensity than the untreated cells.
Ice-cold phosphate-buffered saline-washed cells were lysed in homogenization buffer (10 mM Tris-HCl at pH 7.4, 2 mM EDTA, 1 mM EGTA, 50 mM NaCl, 1% Triton X-100, 50 mM NaF, 20 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 1:100/v:v proteinase inhibitor cocktail) at 4 °C for 30 min. Cell extracts were obtained by ultracentrifugation of cell lysates at 100,000 × g for 30 min at 4 °C. The protein concentration of the cell extract was measured using a protein assay kit, and the extracts were adjusted to 2 mg/mL with homogenization buffer. For immunoblotting analysis, the proteins in the cell extracts were separated by SDS-PAGE and electrotransferred to PVDF membranes using semi-dry blot apparatus (Bio-Rad) at 3 mA per cm2 of gel in transfer buffer (25 mM Tris, pH 8.3, 192 mM glycine, and 20% methanol) at room temperature for 30 min. The free protein-binding sites on the PVDF membrane were saturated by incubation with 5% nonfat milk in Tris-buffered saline with Tween 20 (TTBS) (20 mM Tris at pH 7.4, 0.15 M NaCl, and 0.2% Tween 20) at 25 °C for 2 h. The membrane was then incubated with 0.1 μg/ml primary antibody in TTBS containing 3% nonfat milk at 4 °C overnight and subsequently with secondary antibody conjugated with peroxidase (1:1000) at 25 °C for 1 h. The immunoblots were developed using an enhanced chemiluminescence system, and the luminescent images were captured using a Chemiluminescent Detection System (Chemi Doc. XRS, Bio-Rad).
All data are expressed as means ± SD unless stated otherwise. The differences between groups were calculated using Student’s unpaired t-test. Dose-dependent effects were calculated using simple linear regression. P < 0.05 was regarded as statistically significant. All statistical analyses were performed using SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA).
The inhibitory effect of LCSE on the growth of NSCLC cell lines
The proliferation and colony-forming activities of A549 and NCI-H661 cells were both inhibited by LSCE treatment. As shown in Fig. 1a, the cell survival ratio decreased gradually in both treated cell lines as compared to the untreated cells after 24 h. The sensitivity of the A549 cell line to LCSE was greater than that of NCI-H661 when the dose of LCSE was higher than 25 μg/mL. MRC-5, a human normal lung cell line, was not remarkably influenced by treatment with LCSE at dosage lower than 50 μg/mL. In cancer cell lines, LCSE exhibited approximately 60% growth inhibition with dosage of 100 μg/mL LCSE, while the survival rate decreased to 20% in A549 and 30% in NCI-H661 cells after treatment with 150 μg/mL of LCSE. Growth inhibition of NSCLC cells caused by LCSE treatment was similarly observed in colony-forming activity. As shown in Fig. 1b, the number of colonies gradually reduced as the dosage of LCSE increased. The sensitivities of the two cell lines to LCSE in this assay had similar trend; however, NCI-H661 cells exhibited greater sensitivity than A549 cells in the dosage of 25 μg/mL and 50 μg/mL (P < 0.01). These results demonstrated that LCSE exerted significant inhibition on both the proliferation and colony formation in these two NSCLC cell lines, whereas the cytotoxicity effects in normal lung cell line were relatively low.
Cell-cycle arrest at the G1 or G2/M phase by LCSE treatment
The cell-cycle distribution of LCSE-treated NSCLC cells is shown in Fig. 2a. A549 cells exhibited increasing percentage of G1-phase cells upon LSCE treatment with dosage greater than 12.5 μg/mL. The percentage of total control cells increased from 58.93 to 66.04% as the dosage elevated from 12.5 μg/mL to 50 μg/mL of LCSE. Increasing numbers of cells of G2/M-phase were also noted, from 9.99% of the total control cells to 26.41% in the group of 150 μg/mL LCSE-treated cells. Regarding to the number of S-phase, LCSE-treated cells gradually decreased from 31.07% of the total control cells of 12.5 μg/mL to 11.8% of 150 μg/mL in LCSE-treated cells. NCI-H661 cells also showed a trend of gradual increase in G2/M-phase cells, from 6.01% of the total control cells to 31.54% of 150 μg/mL LCSE-treated cells, but the numbers of cells in G1-phase were reduced. The changes in cell-cycle controlling proteins are shown in Fig. 2b. The levels of cell-cycle controlling proteins, including cyclin D1, E, and B1, decreased gradually, and the level of Kip1/p27 increased after LCSE treatment in A549 cells. The levels of cyclin E and B1 decreased gradually after LCSE treatment in NCI-H661, but the level of cyclin D increased. The elevation of Kip1/p27 level in NCI-H661cells treated with dosage of LCSE greater than 25 μg/mL was also noted. These results indicated that LCSE arrested A549 cells in the G1 phase of the cell cycle and concomitantly inhibited G2/M progression, possibly by suppressing the expressions of cyclin D1, E and B1 and increasing the Kip1/p27 level. The suppression of cyclin with elevation of Kip1/p27 accounted for the LCSE arresting NCI-H661 cell in the G2/M phase of the cell cycle.
Apoptosis induction by LCSE in NSCLC cells
LCSE-treated A549 and NCI-H661 cells were stained with annexin V-FITC or rhodamine 123, and were analyzed by flow cytometry to assess the numbers of apoptotic cells and the mitochondrial integrity. Compared with the control groups, the number of annexin V-positive cells in both LCSE-treated NSCLC cell lines gradually increased according to the dosage elevation. A549 was shown to be less affected by LCSE-induced apoptosis. Contrary to A541, the increasing percentage of annexin V-positive cells from 12% in 25 μg/mL group to 40% in 150 μg/mL group of LCSE treatment, approximately 14% of annexin V-positive cells were observed in 12.5 μg/mL group, and increased to 64% in 150 μg/mLgroup of LCSE-treated NCI-H661 cells (Fig. 3a). To investigate whether the induction of apoptosis by LCSE in NSCLC cells involved the loss of mitochondrial integrity, Δψ m in LCSE-treated cells was analyzed. As shown in Fig. 3b, the percentages of rhodamine 123-negative cells in the control groups were all below 5% of the total cells. After treatment with LCSE, this percentage increased significantly (P < 0.05), from 13% in the group of 12.5 μg/mL to around 80% in the 150 μg/mL group of LCSE-treated NCI-H661 cells. The percentage of rhodamine 123-negative A549 cells also increased from around10% in 25 μg/mL group to around 25% in the 150 μg/mL LCSE treatment. Immunoblotting analysis revealed that the levels of pro-caspase 8, 9 and 3 were decreased when treated with dosage greater than 25 μg/mL LCSE, while the caspase 3 substrate PARP showed increasing levels of cleaved fragments under the same treatment conditions in A549 cells (Fig. 3b). The level of apoptosis-promoting protein Bax was increased in A549 cells when treated with greater than 50 μg/mL LCSE. However, the level of anti-apoptosis protein Bcl-2 was sustained under LCSE treatment in A549 cells. The levels of pro-caspase 8, 9 and 3 were gradually decreased and the caspase 3 substrate PARP showed increasing levels of cleaved fragments in LCSE treated NCI-H661 cells that were similar to A549 cells with dosage greater than 12.5 μg/mL. The level of apoptosis-promoting protein Bax was increased in NCI-H661 cells under treatment with dosage greater than 25 μg/mL LCSE, while the level of anti-apoptosis protein Bcl-2 was decreased under treatment with dosage greater than 50 μg/mL LCSE in NCI-H661.
LCSE disturbed EGFR signaling in NSCLC cells
EGFR protein and its phosphorylated tyrosine level were gradually decreased in A549 cells treated with dosage greater than 25 μg/mL LCSE (Fig. 4). The expression in the protein level of EGFR was unremarkable, while the phosphor-EGFR level was decreased in cells treated with dosage greater than 25 μg/mL of LCSE in NCI-H661 cells. Both the protein level and phosphorylation level of EGFR downstream signal protein Akt were decreased in the two LCSE-treated NSCLC cell lines. The protein levels of Erk-1 and −2 were not altered under LCSE treatment below 50 μg/mL but were slightly decreased under treatment with greater than 100 μg/mL of LCSE in A549 cells. The levels of phosphor-Erk-1 and −2 were decreased in cells treated with dosage greater than 25 μg/mL of LCSE in A549 cells. The levels of these two Erk proteins were slightly increased when treated with dosage below 25 μg/mL of LCSE and were decreased under the dosage greater than 50 μg/mL in NCI-H661. The levels of phosphor-Erk-1 and −2 were significantly decreased under treatment with dosage greater than 12.5 μg/mL LCSE and were undetectable under treatment with dosage of 150 μg/mL LCSE in NCI-H661 cells.
Previous studies have revealed inhibitory effects of LCSE on the growth of several types of malignant cells, including lung cancer [8, 11]. However, the exact mechanisms responsible for the anti-cancer activity of LCSE are still not fully understood. In this study, we comprehensively investigated the possible cellular and molecular mechanisms of the effects of LCSE in two NSCLC cancer cell lines, A549 and NCI-H661. The results demonstrated that LCSE suppressed the EGFR protein and its phosphorylation levels, then downregulated the downstream signaling, such as Erk-1/-2 and Akt signaling. These effects resulted in the suppression of some cyclins, elevation of the expressions of Kip1/p27 and Bax protein, and activation of caspase-8, −9 and −3, which induced cell-cycle arrest and apoptosis in NSCLC cells.
EGFR is the predominant target of therapy in NSCLC, as this proto-oncogene is always overexpressed and plays a key role in the growth of NSCLC . Activated EGFR stimulates its two major downstream signaling pathways, Ras and phosphatidyl inositol 3 kinase (PI3K), then activates Erk-1/-2 and Akt signaling. Erk-1/-2 translocates to the nucleus and triggers expression of cell-cycle-controlling proteins. Akt could phosphorylate Bad and dissociate Bad with Bcl-2, which suppresses the activity of Bax and leads to the avoidance of apoptosis in cancer cells . In this study, we demonstrated the elevation of the number of apoptotic cells, and the reduction in mitochondria membrane potential in both treated A549 and NCI-H661cells with LCSE. The expression of Bax protein was upregulated in both cells, and Bcl-2 was downregulated in NCI-H661 cells. These results indicated that the inhibitory effect of LCSE on EGFR signaling directly effected the Akt activity, then triggered an intrinsic apoptosis mechanism to induce apoptotic death in both NSCLC cells. A previous report indicated that LCSE also triggers the other extrinsic apoptosis pathway to induce apoptosis . Our results appeared to support this viewpoint, with the level of procaspase 8 being gradually decreased after LCSE treatment in the NSCLC cells. From our results and those of other studies, we can conclude that LCSE-induced apoptotic death in NSCLC cells arises from triggering both the intrinsic and extrinsic apoptosis pathways.
Although LCSE induced apoptosis in both tested NSCLC cells, the sensitivity differed between the two cells. NCI-H661 cells were more sensitive than A549 cells in the treatment with LCSE which was demonstrated by the number of annexin V-positive cells, the mitochondria potential loss and the Bax and Bcl-2 protein levels. As shown in Fig. 3, the number of annexin V-positive cells and the mitochondria membrane potential loss of cells were elevated more than 60% of the total NCI-H661 cells under LCSE treatment with dosage of 100 and 150 μg/mL. In the A549 cells, only less than 40% of the total number of cells underwent apoptosis. Although the Bax level in LCSE-treated cells was elevated, the Bcl-2 level in NCI-H661 cells was dramatically diminished under dosage more than 50 μg/mL LCSE treatment, whereas the level of Bcl-2 in A549 cells was sustained. The balance of Bax and Bcl-2 in cells plays a pivotal role in the regulation of apoptosis in LCSE-treated cancer cells which was highlighted in our previous report and other studies [8, 11, 12]. LCSE-treated NCI-H661 cells contained a greater number of apoptotic cells indicating that the mechanism of the growth inhibition effect of LCSE in the cells is mitochondria-mediated apoptosis, which mainly arised from elevation of the Bax level and downregulation of the Bcl-2 level.
Our previous study revealed that LCSE arrested cancer cells in the G2/M phase by reducing the expression of cyclin B1 . In this study, we further confirmed this effect in the two tested NSCLC cells. The number of NCI-H661 cells in the G2/M phase was elevated under treatment with LCSE with dosage more than 12.5 μg/mL. However, the change of G2/M phase only gradually increased to a quarter of A549 cells under LCSE treament with dosage of 150 μg/mL. These results suggest the effect of cell cycle arrest in G2/M phase was more predominant in NCI-H661 cells. Previous studies revealed that Erk activity is an important factor in G2/M-phase progression in the cell cycle, and the M phase is nearly twice as long in mammalian cultured cells when Erk is inhibited during G2/M transition [17, 18]. In the report of Lin et al., they indicated that downregulation of the EGFR/Erk/c-fos signaling pathway can inhibit the COX-2 level and activate Kip1/p27 to induce G2/M cell-cycle arrest in oral carcinoma cells . Another study revealed that EGF could activate Akt and triggered CK2a and HDAC2 expression; whereas inhibition of Akt or CK2a and concomitant suppression of the expressions of CDC25c and cyclin B would arrest cells in the G2/M phase . Our results proved that LCSE inhibited the activities of EGFR, Erk-1/-2 and Akt and concomitantly suppressed the level of cyclin B; therefore, indicating that the LCSE-induced G2/M-phase arrest might arise from the reduction of EGFR signaling and control of cyclin B in NSCLC cells.
However, LCSE also disturbed the process of the G1 phase progression in A549 cells, including suppression of the levels of cyclin D1 and E and elevation of the level of Kip1/p27. Our results confirmed that LCSE-treated A549 cells expressed a greater number of G1-phase cells as compared to control cells. Recent studies proved that suppression of the EGFR/Ras/MEK/Erk and PI-3 K/Akt pathways could lead to cell-cycle arrest in the G1 phase [21, 22], mainly through upregulation of Kip/p27 and downregulation of cyclin E [23, 24]. Earlier reports also pointed out that Erk regulates cyclin D1 transcription and cyclin D1/CDKs assembly and triggers G1 to S transition in the cell cycle [25–29]. Since LCSE reduced EGFR signaling, downregulated Erk-1/-2 and Akt, and arrested the G1 phase in A549 cells, our results indicated a possible novel role of LCSE in the control of lung adenocarcinoma cells in terms of cell-cycle progression from the G1 phase to the S phase. However, the effect of G1 phase arrest was not fully apparent in LCSE-treated NCI-H661 cells, in which cyclin D1 was elevated under LCSE treatment. The reasons for these results need further investigation.
Our results revealed that LCSE inhibited EGFR activity and protein expression in two NSCLC cells, concomitantly reduced the downstream Akt and Erk-1/-2 signaling, then downregulated Bcl-2, cyclin B1 and cyclin E and elevated Kip1/p27 and Bax. In addition to growth inhibition in both cell lines, these events resulted in cell arrest at the G2/M phase of the cell cycle and lead to apoptotic death. This study was the first to describe the detailed mechanisms of LCSE in NSCLC and suggested LCSE may be a novel herbal agent that acts through the inhibition of EGFR signaling to induce cytotoxicity in NSCLC. It would be intriguing to examine whether the findings of this study could be replicated in an animal model of NSCLC and further investigation is warranted.
Litchi seed extract
Non-small cell lung cancer
Polyacrylamide gel electrophoresis
Sodium dodecyl sulfate
Tyrosine kinase inhibitor
- Δφ m :
Mitochondria membrane potential
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The authors appreciated the technical support from Mr. Shang-Hao Liu and Miss Ying-Hua Hsu.
This work was supported by a grant from Cheng-Ching Hospital of Taiwan (CH103A002).
Availability of data and material
The datasets during and/or analysed during the current study available from the corresponding author on reasonable request.
YCC provided the grant and prepared the draft of the manuscript. CHC and YTT performed all experiments and collected all of the results and data analysis. CCL and JCC analyzed and made the quality assurance of each batch of the LCSE. TYK, CCH and CHC performed the statistical analysis of the data and reviewed the draft of manuscript. CPH, as the corresponding author, designed the scheme and all of the experiments as well as corrected the draft of manuscript. All authors have read and approved the final manuscript.
The authors declare that they have no competing interest.
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