|Year : 2016 | Volume
| Issue : 4 | Page : 1284-1290
Enhanced cytotoxic activity of endophytic bacterial extracts from Adhatoda beddomei leaves in A549 lung cancer cell lines
Y Swarnalatha, Bhaswti Saha
Department of Biotechnology, Sathyabama University, Chennai, Tamil Nadu, India
|Date of Web Publication||7-Feb-2017|
Department of Biotechnology, Sathyabama University, Chennai - 600 119, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Aim of the Study: The current study is aimed to isolate and study the efficacy of the anticancer activity of endophytic bacteria from adhatoda beddomei leaves. Endophytic bacteria, microorganisms can found in the plant tissues, like leaves, branches and roots and able to produce various novel secondary metabolites for the medicinal applications.
Methodology: Endophytic bacteria were isolated from the leaves of the adhatoda beddomei leaves. The extract from the culture was tested for the cytotoxicity in A549 cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assay, dual staining and nuclear staining.
Results: The expression of the apoptotic and proliferative genes were assessed with the reverse transcriptase polymerase chain reaction (RT-PCR) comparing with the control gene. The inhibitory concentration (IC50) of the bacterial extract was found to be 43.97 μg/ml. With the dual staining the apoptotic cell percentage was significantly increased (P < 0.001) and with the 40μg/ml and 80μg/ml concentration the apoptotic percentages observed as 67% and 89% respectively. Similar concentrations were used for the nuclear fragmentation (PI) and the cell cycle analysis (FACS) using WinMDI 2.9 software. During cell cycle analysis the accumulation of the cells at G0-G1 stage was observed with increasing concentrations of the chi-alg encophytic bacterial extract nanoparticles. Finally the proapoptotic and proliferative gene expression for the bax, Bcl-2 and caspase was significantly regulated (P < 0.01; P < 0.05). The Bax and Caspase were up-regulated and Bcl-2 was down regulated.
Conclusion: The results conclude that enophytic bacterial extract possess good cytotoxic activity.
Keywords: Adhatoda beddomei, cytotoxicity, endophytic bacteria, nuclear fragmentation, reverse transcription polymerase chain reaction
|How to cite this article:|
Swarnalatha Y, Saha B. Enhanced cytotoxic activity of endophytic bacterial extracts from Adhatoda beddomei leaves in A549 lung cancer cell lines. J Can Res Ther 2016;12:1284-90
|How to cite this URL:|
Swarnalatha Y, Saha B. Enhanced cytotoxic activity of endophytic bacterial extracts from Adhatoda beddomei leaves in A549 lung cancer cell lines. J Can Res Ther [serial online] 2016 [cited 2021 May 7];12:1284-90. Available from: https://www.cancerjournal.net/text.asp?2016/12/4/1284/161928
| > Introduction|| |
Cancer causes serious problem which leads to morbidity and mortality and creates worldwide problem in public health. Effective anticancer compounds are not well known. Plant-based compounds are better curative compounds than synthetic compounds. In spite endophytes are the organisms which are found inhabiting in the plant tissues in various parts  and potential source to produce various bioactive compounds. Endophytes are of two different types: Fungi and bacteria. The anticancer drug paclitaxol is a known anticancer drug which was isolated from endophytic fungi. In the year of 2007 Newman and Cragg listed out the various drugs approved from 1981 to 2006, out of which maximum number of natural agents were synthesized from endophytes. Endophytes in pharmaceutical industries applications provides a cost effective drug production, and also helps in the conservation of biodiversity and drug resistance as they are an alternate source of drugs. The anticancer properties of several secondary metabolites from endophytes have been investigated recently.
Adhatoda beddomei is a shrub; the leaves of the plant are lance-shaped with 10-15 cm in length by four wide. The leaves are alternatively arranged with smooth edged, short petioles. The herb is distributed throughout India and commonly known as Vasa or vasaka. The isolated alkaloids vasicine and theophylline have good respiratory stimulation effect. The cytotoxic activity of the endophytic fungi from the Adhatoda beddomei was proved by Prabavathy 2013. However, the anticancer activity of endophytic bacterial extract from this plant has not been proved yet; thus, the current search was an attempt to identify the anticancer activity of the endophytic bacterial extract.
| > Materials and Methods|| |
Endophytic bacterial source
The healthy leaf material was collected from Siddha Medicinal Plants Garden, Mettur. The leaf samples were collected in sterile plastic bags and transported to laboratory in October, 2014. The obtained leaves were stored at 4°C for future purpose. These leaves were used for isolation of endophytic bacteria.
Isolation of endophytic bacteria and preparation of extraction
The endophytic bacteria were isolated according to the;, with minor modifications. The endophytic bacteria was isolated and identified as Lactobacillus spp, the information was published earlier by the author. The pure culture obtained were subcultured on LB agar plates for 24 h, then subcultured on to Luria bertani in 1l Erlenmeyer flask and incubated in shaker incubator at 37°C for 48h. After incubation, the culture broth was subjected to sonication for 1h to break the cells and chloroform was used for extraction in separating funnel by shaking vigorously for 30 min. The mixture was allowed to settle until two distinct layersappeared, the upper solvent layer and lower aqueous layer. The upper solvent layer was separated and evaporated in rotary evaporator and the crude extract was obtained. The obtained extract was dissolved in dimethyl sulfoxide (DMSO) and preserved in refrigerator. This crude extract was used for further experimental purpose. The bioactive compounds were analyzed and identified, the identified bioactive compound details were published by the author.
Preparation of bank chitosan-alginate nanoparticles
Sodium alginate (1%) and sodium silicate (1.5%) were prepared by dissolving the chemicals in distilled water. Chitosan (1%) was dissolved in 2N acetic acid. Chitosan-alginate nanoparticles were formulated based on the ionotropic gelation. The pH was adjusted to 7. The white colored hydrogels formed was observed, and these were filtered out and washed with the help of distilled water. These hydrogels were subjected to lyophilization to obtain in dry form 
Preparation of endophytic bacterial extract loaded chitosan-alginate nanoparticles
Ten milliliters of endophytic bacterial extract was incorporated into 10 ml of chitosan solution. It was then added to the sodium-alginate solution and then the subsequent steps were similar to that of the preparation of the blank chitosan-alginate nanoparticles.
Scanning electron microscope analysis
The topographical analysis and the surface characteristics of the beads were obtained by analyzing the samples under a Scanning electron microscope (SEM) (Carl Zeiss SUPRA-55).
A549 human lung adenocarcinoma cell line was procured from National Centre for Cell Science (NCCS), Pune, India with the passage number of 29. Cells were maintained in Dulbecco's minimum essential media (DMEM) supplemented with 10% fetal bovine serum (FBS), with 100 unit/ml penicillin and 100 μg/ml streptomycin. Cells were cultured in a humidified atmosphere with 5% CO2 at 37°C. Cells were grown in 75cm 2 culture flask and after a few passages, cells were seeded for experiments. The experiments were done at 70-80% confluence. Upon reaching confluence, cells were detached using 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) solution.
Cytotoxicity (MTT assay)
The cytotoxicity of the endophytic bacterial extracts was assessed using MMT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)) according to Safadi et al., 2003. The plates were incubated for 24h at 37°C and then spectrophotometrical absorbance of the purple blue formazan dye was measured using an ELISA reader (BIORAD) at 570 nm. Optical density of each sample was compared with control optical density and graphs were plotted. The results were expressed as mean half maximal inhibitory concentration (IC50) of three independent experiments (± standard deviation).
Cell morphological assay using dual staining (EB/AO)
Cellular morphology was assessed using EB/AO stain according to the method of Gohel et al., 1999. The cell culture was maintained to get 70–80% confluent of cells at 37°C in humidified CO2 incubator. Then chitosan-alginate blank and chitosan-alginate encapsulated tests was prepared in a concentration of 20 and40 µg/ml and 40 and 80µg/ml (selected based on the IC50 concentration) treated for 24h. The culture medium was aspirated from each well and cells were gently rinsed twice with phosphate-buffered saline (PBS) at room temperature. Then equal volumes of cells from control and drug treated were mixed with 100 μl of dye mixture (1:1) of ethidium bromide and acridine orange (AO) and viewed immediately under Nikon inverted fluorescence microscope (Ti series) at ×10 magnification. A minimum of 300 cells was counted in each sample at two different fields. The percentage of apoptotic cells was determined by % of apoptotic cells = ((total number of apoptotic cells/total number of cells counted) ×100).
Assessment of nuclear morphology after propidium iodide (PI) staining
There will be significant morphological changes in the nuclear chromatin of the cell undergoing apoptosis. These morphological changes can be assessed using PI staining. The staining was carried out by the method of Chandramohan et al., 2007. A549 cells were plated at a density of 5 × 104 in six-well plates. They were allowed to grow at 37°C in a humidified CO2 incubator until they were 70–80% confluent. Then cells were treated with 40 and 80µg/ml of chitosan-alginate encapsulated endophytic bacterial extract for 24h. The cells were harvested from the culture medium and rinsed gently with PBS at room temperature, then fixed in methanol: acetic acid (3:1v/v) for 10 min, and nuclear morphological changes were observed under flow cytometerafter staining with50μg/ml PI for 20 min. The percentage of apoptotic cells was calculated; at least, 1 × 103 cells were counted for assessing apoptotic cell death. This percentage of calculation of apoptotic cells was done according to the increase in the uptake of the PI stain between the control and the chitosan-alginate encapsulated endophytic bacterial extract.
Cell cycle analysis
The cell cycle distribution was studied by preparing the A549 cells in a concentration of (1 × 105 cells/ml). The cells were treated with chitosan-alginate encapsulated endophytic bacterial extract in a concentration of 40 and 80µg/ml and incubated for 24h. The treated cells were harvested, washed with PBS, and fixed in 75% ethanol at 4°C overnight. Then collected and washed twice with cold PBS, cells were suspended in PBS containing 40μg/ml PI and 0.1mg/ml ribonuclease (RNase) A followed by shaking at 37°C for 30 min. The stained cells were analyzed with flow cytometer (Becton-Dickinson San Jose, CA, USA) and the data were consequently calculated using WinMDI 2.9 software (TSRI, La Jolla, CA, USA)
Analysis of apoptotic markers using reverse transcription polymerase chain reaction (RT-PCR)
Human lung adenocarcinoma cancer A549 cells were cultured in six-well plates and exposed to 80 and 160μg/ml chitosan-alginate encapsulated endophytic bacterial extract for 24 h. At the end of exposure, total RNA was extracted by Trizol reagent according to the standard protocol. Concentration of the extracted RNA was determined and the integrity of RNA was visualized on a 1% agarose gel using a gel documentation system (BioRad, Hercules, and CA). The first strand of Complimentary DNA (cDNA) was synthesized from 1 μg of total RNA by reverse transcriptase using M-MLV (Promega, Madison, WI) and oligo (dT) primers (Promega) according to the manufacturer's protocol. Quantitative real-time PCR was performed by QuantiTect SYBR Green PCR kit (Qiagen) using an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA). Two microliters of template cDNA was added to the final volume of 20 μL of reaction mixture. Real-time PCR cycle parameters included 10 min at 95°C followed by 40 cycles involving denaturation at 95°C for 15 s, annealing at 60°C for 20 s, and elongation at 72°C for 20 s. The sequences of the specific sets of primer for Bax, Bcl-2, caspase-3, and β-actin used in this study were taken from literatures. Expressions of selected genes were normalized to the β-actin gene, which was used as an internal housekeeping control. All the real-time PCR experiments were performed in duplicate, and data were expressed as the mean of at least two independent experiments.
Data were expressed mean ± standard error of the mean by Tukey's test to determine the significance of differences between groups. A P value lower than 0.05, 0.01, and/or 0.001 was considered to be significant.
| > Results|| |
The endophytic bacteria isolated and identified from Adhatodabeddomei as Lactobacilli sppwas submitted to GenBank and the accession number provided was KJ680325. Dose-dependent inhibition of the proliferation of the chitosan-alginate encapsulated endophytic Lactobacilli spp in A549 cancer cells.
Determination of SEM
The scanning electron microscope revealed the formation of the blank and encapsulated chit-alg nanoparticles [Figure 1] of the endophytic bacterial endophytic extract. [Figure 1]a indicates the chi-alg blank nanoparticles with flat surface. [Figure 1]b and [Figure 1]c indicates the chi-alg nanoparticles encapsulated endophytic bacterial extract with large and bulged surface area.
|Figure 1: (a) SEM of blank chitosan-alginate nanoparticles. (b and c) Endophytic bacterial extract encapsulation with chitosan-alginate nanoparticles|
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The effect of endophytic Lactobacilli spp chitosan-alginate particles anticancer activity was assessed using MTT. After 24h treatment with various concentrations of test particles, percentage of inhibition was recorded. Maximum inhibition was found to be 90% at a concentration of 200 µg/ml. The IC50 was calculated by linear regression analysis. The IC50 of the particles was 43.97 µg/ml. Hence, further assays were carried out with 40 and 80 µg/ml [Table 1].
|Table 1: Cytotoxicity of nano blank and nano encapsulated endophytic bacterial extract against A549 human lung adenocarcinoma cell line|
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Dual staining to assess the morphological changes of the A549 cancer cell
The morphological variations are evaluated using dual staining. The cellular morphology changes can be observed distinctively for the live cells and apoptotic cells. There is large distinction in the intake of the stain between the live, early apoptotic cells, and late apoptotic cells. Live cells uptake the AO and emits the green fluorescence. Whereas, the early apoptotic cells with fragmented DNA also uptake the AO and emit intense green color; and the late apoptotic and necrotic cells were fragmented and stained orange with red colored nuclei [Figure 2]a,[Figure 2]b,[Figure 2]c. The cellular morphology changes can be observed distinctively for the live cells and apoptotic cells. The data clearly indicates that with increase in the concentration of particles there is tremendous decrease in the number of viable cells. The percentage of apoptotic cells after treatment with 40 and 80µg/ml of drug was drastically increased (P < 0.001) to 67 and 89%, respectively [Figure 3].
|Figure 2: Acridine orange/ethidium bromide staining of nanoencapsulated endophytic bacterial extract|
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|Figure 3: Cytotoxicity of nanoblank and nanoencapsulatedendophytic bacterial extract against A549 human lung cancer cell line|
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Effect of chitosan-alginate encapsulated endophytic bacterial extract on nuclear fragmentation
Apoptosis can further confirmed by analyzing the nuclear morphology stained with PI stain. The PI stain is impermeant to the live cells, but the dead cells can easily uptake the dye and stain themselves in to red fluorescence by binding tightly to the nucleic acids. The characteristic changes are due to the nuclear fragmentation of nuclei in treated A549 cells; whereas, the untreated control cells did not show any nuclear fragmentation [Figure 4] The nucleolus disintegrates; nuclear membrane develops deep invaginations and, ultimately, the nucleus fragments into dense granular particles (apoptotic bodies) in a dose dependence manner with 40 and 80μg/ml of the extract increased enormously (P < 0.001) to 62 and 90%, respectively [Figure 5]a,[Figure 5]b,[Figure 5]c.
|Figure 4: Bar graph showing the percentage number of apoptotic cells in chitosan-alginate blank nanoparticles and chitosan-alginate encapsulated endophytic bacterial extract|
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|Figure 5: Propidium iodide staining assay of nanoencapsulatedendophytic bacterial extract|
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Cell cycle analysis using fluorescence activated cell sorting
Cell cycle analysis confirms whether the cause of A549 cell death induced by chitosan-alginate encapsulated endophytic bacterial extract was apoptosis, Cell cycle analysis confirms whether the cause of A549 cell death induced by chi-alg encapsulated endophytic bacterial extract was apoptotic cell death, using fluorescence activated cell sorting (FACS). Incubation of fixed and permeabilized cells with fluorochrome PI results in quantitative PI binding with total cellular DNA, and the fluorescence intensity of PI-labeled cells was proportional to DNA contents in a dose-dependent manner. At lower concentration (40µg/ml), the chitosan-alginate encapsulated endophytic bacterial extract showed arrest at sub-G0/G1 with 15% cells accumulated. At higher concentration (80µg/ml), it showed an increased cell population at sub G0–G1 with 30% and concomitant decrease in the other phases of cell cycle. Sub G0–G1 phase is the apoptotic phase, hence from the results it is confirmed that treatment with chitosan-alginate encapsulated endophytic bacterial extract induced A549 cell apoptosis maximally. In double variable flow cytometry scatter plot, R1 quadrant (fluorescein isothiocyanate (FITC)-/PI-) indicates living cells in [Figure 6], which represents the control group. The [Figure 7] and [Figure 8] shows that the chitosan-alginate encapsulated endophytic bacterial extract treated cells; the R1quadrant stands for living cells and the scatters (FITC+/PI+) out of the quadrant indicates population of the cells in apoptotic phase.
|Figure 6: The percentage of apoptotic nuclei of the control and chitosan-alginate nanoparticles. EB = Endophytic bacteria|
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|Figure 7: FACs analsis -showing the percentage of apoptotic nuclei of the control and chi-alg nanoparticles|
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|Figure 8: FACs analysis- Cell Cycle arrest induced by different concentrations of chi-alg nanoparticles endophytic bacterial extract|
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Expression of apoptotic related proteins in A549 cells
The apoptotic process is controlled by several gene products. The determination of the effectiveness of chitosan-alginate encapsulated endophytic bacterial extract nanoparticles on the expression of apoptotic genes was analyzed with estimation of mRNA using quantitative real-time PCR (Bax, Bcl-2, β-actin, and caspase). The cellular lysate extracts were analyzed by western blotting to determine their expression level in dose-dependent manner. [Figure 10] reveals that the expression of proapoptotic and antiapoptotic genes was in a dose-dependent manner. [Figure 9] indicates mode of expression of the Bax, Bcl-2, and caspase; from this it revealed that the expression of theproapoptotic gene, Bax, and caspase-3 were significantly upregulated; whereas the antiapoptotic protein was significantly downregulated (P < 0.01, P < 0.05). β-actin was used as housekeeping gene or endogenous control.
|Figure 9: Cell cycle arrest induced by different concentrations of nano encapsulated endophytic bacterial extract|
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| > Discussion|| |
From the current study the data indicates that the chitosan-alginate encapsulated endophytic bacterial extract induced permanent inhibitory result on the proliferation in A549 cells. The cells were exposed to drug in dose- and time-dependent manner to check the ability of the drug to inhibit the proliferation for 12-48h. The drug showed an irreversible effect on the proliferation of the cells; there was an apparent decrease in the number of cells due to induction of apoptosis in the A549 cells.
The current finding proves that the chitosan-alginate encapsulated endophytic bacterial extract possess the anticancer activity. This apoptotic nature was characterized with observation of the morphological changes, such as nuclear condensation and apoptotic bodies formed [Figure 2], this was confirmed using PI staining.
There are group of genes involved in the regulation of the apoptosis which includes Bcl-2 and Bax. Bcl-2 is an antiapoptotic gene and protects cells against the apoptosis in a variety of experimental systems. Various types of stimuli and anticancer drugs suppress the expression of Bcl-2 to promote apoptosis. Bax and caspase-3 exhibits their apoptotic effect by binding to Bcl-2 protein family and also prevents the activation of cytochrome c. In our study, the levels of the antiapoptotic factor Bcl-2 protein expression were decreased after treating with the drug. Whereas proapoptotic genes like Bax and caspase-3 protein expression was unregulated. Actin is an important filament in the plasma membraneassociated with cytoskeleton, and is a major component in the determination of the cell polarity, shape, and mechanical properties of the plasma membrane. There are several reports exerting the relationship between the apoptosis and actinfilament disruption. But the levels of actin will not be altered with the condition of the cell, so the β-actin was used as control. These β-actin genes are the house keeping genes that are widely used for gene expression normalization for RT-PCR. There are experiments which revealed that their expression may be dependent on the type of tissue, developmental stage, and the experimental conditions followed.
Finally in our study it has been concluded that, the chitosan-alginate encapsulated endophytic bacterial extract significantly showed anticancer activity in A549 cells in dose- and time-dependent manner by inducing apoptosis, and that the progression of apoptosis was associated with the disruption of actin fibers. We also confirmed that the Bcl-2 protein was downregulated and Bax and caspase-3 were upregulated, but there is no change in the expression of the β-actin by chi-alg encapsulated endophytic bacterial extract treatment.
| > References|| |
Wilson D. Ecology of woody plant endophytes, in Microbial endophytes. NY, USA: Mercel Dekker; 2000. p. 389-420.
Newman DJ, Cragg GM. Natural products as sources of new drugs over the last 25 years. J Nat Prod 2007;70:461-77. Epub 2007 Feb 20.
Avula B, Begum S, Ahmed S, Choudhary MI, Khan IA, Khan IA. Quantitative determination of vasicine and vasicinone in Adhatoda vasica by high performance capillary electrophoresis. Die Pharmazie – An International Journal of Pharmaceutical Sciences 2008;63:20-22.
Prabavathy D, Valli Nachiyar C. Cytotoxic potential and phytochemical analysis of justicia beddomei and its endophytic aspergillus SP. Asian Journal of Pharmaceutical and Clinical Research 2013;6:159-61.
Pal Arundhati, Paul AK. Bacterial Endophytes of the medicinal herb Hygrophia spinosa T. Anders and their Antimicrobial activity. British Journal of Pharmaceutical Research 2013;l:795-806.
Bhore Subhash J, Nithya Ravichantar, Loh Chye Ying. Screening of endophytic bacteria isolated from leaves of Sambung Nyawa [Gynura procumbens (Lour.) Merr.] for cytokinin-like compounds. Bioinformation 2011;5:191-97.
Swarnalatha, Bhaswati Saha. Isolation, characterization and antibacterial activity of endophytic bacteria from adhathoda beddomei. Int J Pharm Bio Sci 2014;5:305-10.
Paul Catalin Bacalaureat, Ecaterina Andronescub, Alexandru Mihai Grumezescub, Anton Ficai B, Keng Shiang Huangc, Chih Hui Yangd, et al
. Fabrication, Characterization and in-vitro
Profile based interaction with Eukaryotic and Prokaryotic cells of Alginate Chitosan Silica Biocomposite. International Journal Of Pharmaceutics 2012;4:555-61.
Safadi FF, Xu J, Smock SL, Kanaan RA, Selim AH, Odgren PR,et al
. Expression of connective tissue growth factor in bone: its role in osteoblast proliferation and differentiation in vitro
and bone formation in vivo
. J Cell Physiol 2003;196,51-62.
Gohel A, McCarthy MB, Gronowicz G. Estrogen prevents glucocorticoid-induced apoptosis in osteoblasts in vivo
and in vitro
. Endocrinology 1999;140:5339-47.
Chandramohan KVP, Gunasekaran P, Varalakshmi E, Hara. Y, Nagini S. In vitro
evaluation of the anticancer effect of lactoferrin and tea polyphenol combination on oral carcinoma cells. Cell Biol Int 2007;31:599-608.
Tu LC, Chou CK, Chen CY, Chang YT, Shen YC, Yeh SF. Characterization of the cytotoxic mechanism of Mana-Hox, an analog of manzamine pomegranate seeds. Biochimica et Biophysica Acta 2004;1672:148-56.
Fisher TC, Milner AE, Gregory CD, Jackman AL, Aherne GW, Hartley JA, et al
. Bcl-2 modulation of apoptosis induced by anticancer drugs: resistance to thymidylate stress is independent of classical resistance pathways. Cancer Res 1993;53:3321-26.
QingYong Chen, Li Jun Wu, Yu Quan Wu, Guo Hua Lu, Zhong Yong Jiang, Jian Wei Zhan, et al
. Molecular mechanism of trifluoperazine induces apoptosis in human A549 lung adenocarcinoma cell lines. Molecular Medicine Reports 2009;2:811-17.
Lee A, Fischer RS, Fowler VM. Stabilization and remodeling of the membrane skeleton during lens fiber cell differentiation and maturation. Dev Dyn 2000;271:257-70.
Van de Water B, Kruidering M, Nagelkerke JF. F-actin dis-organization in apoptotic cell death of cultured rat renal proximal tubular cells. Am J Physiol 1996;270:F593-603.
Juntang Lin, Christoph Redies. Histological evidence: Housekeeping genes beta-actin and GAPDH are of limited value for normalization of gene expression. Development Genes and Evolution 2012;222:369-76.
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