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Journal of Chinese Integrative Medicine ›› 2012, Vol. 10 ›› Issue (9): 1025-1038.doi: 10.3736/jcim20120912

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Chelidonine isolated from ethanolic extract of Chelidonium majus promotes apoptosis in HeLa cells through p38-p53 and PI3K/AKT signalling pathways

Avijit Paul1, Kausik Bishayee1, Samrat Ghosh1, Avinaba Mukherjee1, Sourav Sikdar1, Debrup Chakraborty1, Naoual Boujedaini2, Khuda-Bukhsh Anisur Rahman1()   

  1. 1. Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, India
    2. Boiron Laboratory, Lyon, France
  • Received:2012-02-16 Accepted:2012-04-10 Online:2012-09-20 Published:2018-09-15
  • Contact: Rahman Khuda-Bukhsh Anisur E-mail:prof_arkb@yahoo.co.in

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Objective: To evaluate the role of chelidonine isolated from ethanolic extract of Chelidonium majus in inducing apoptosis in HeLa cells and to assess the main signalling pathways involved.

Methods: Cells were initially treated with different concentrations of chelidonine for 48 h and the median lethal dose (LD50) value was selected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Morphological analysis of nuclear condensation and DNA damage and fragmentation were measured by 4′,6-diamidino-2-phenylindole staining and comet assay. Further, reactive oxygen species (ROS) generation, cell cycle arrest and change in mitochondrial membrane potential were also examined and analyzed by flow cytometry. Evaluation of interaction of drug with CT DNA was investigated by circular dichroism (CD) spectral analysis to find any possible drug-CT DNA interaction. The mRNA and protein expressions of major signal proteins like p38, p53, protein kinase B (AKT), phosphatidylinositol 3-kinases (PI3K), Janus kinase 3 (JAK3), signal transducer and activator of transcription 3 (STAT3) and E6 and E7 oncoproteins as well as the pro-apoptotic genes and antiapoptotic genes were also estimated by reverse transcriptase-polymerase chain reaction and Western blotting.

Results: Based on LD50 value (30 μg/mL) of chelidonine, three doses were selected, namely, 22.5 μg/mL (D1), 30.0 μg/mL (D2) and 37.5 μg/mL (D3). Results showed that chelidonine inhibited proliferation and induced apoptosis in HeLa cells through generation of ROS, cell cycle arrest at sub-G1 and G0/G1 stage, change in mitochondrial membrane potential and fragmentation of DNA. Results of CD spectra showed effective interaction between chelidonine and calf thymus DNA. Studies of signalling pathway revealed that chelidonine could efficiently induce apoptosis through up-regulation of expressions of p38, p53 and other pro-apoptotic genes and down-regulation of expressions of AKT, PI3K, JAK3, STAT3, E6, E7 and other antiapoptotic genes.

Conclusion: Chelidonine isolated from Chelidonium majus efficiently induced apoptosis in HeLa cells through possible alteration of p38-p53 and AKT/PI3 kinase signalling pathways.

Key words: chelidonine, Chelidonium majus, antineoplastic agents, phytogenic, signal transducing, apoptosis, HeLa cells

"

Primer name Primer sequences
Caspase 3 Forward: 5′-AGGGGTCATTTATGGGACA-3′
Reverse: 5′-TACACGGGATCTGTTTCTTTG-3′
PARP-1 Forward: 5′-GATTCCCCATCTCTTTCTTTACACA -3′
Reverse: 5′-GGGCAATAGTCATCACAGACGTT-3′
PI3K Forward: 5′-TTAAACGCGAAGGCAACGA-3′
Reverse: 5′-CAGTCTCCTCCTGCTGCTGAT-3′
AKT Forward: 5′-CCTGGACTACCTGCACTCTCGGAA-3′
Reverse: 5′-TTGCTTTCAGGGCTGCTCAAGAAGG-3′
Bax Forward: 5′-AGTAACATGGAGCTGCAGAGG-3′
Reverse: 5′-ATGGTTCTGATCAGTTCCGG-3′
Bcl-2 Forward: 5′-GTGACTTCCGATCAGGAAGG-3′
Reverse: 5′-CTTCCAGACATTCGGAGACC-3′
ICAD Forward: 5′-ATGGTCAAGCCCAGACAGAG-3′
Reverse: 5′-CGTGTTTTCAACATTTAATGCAA-3′
p53 Forward: 5′-GGAAATTTGTATCCCGAGTATCTG -3′
Reverse: 5′-GTCTTCCAGTGTGATGATGGTAA-3′
GAPDH Forward: 5′-CCCACTAACATCAAATGGGG-3′
Reverse: 5′-CCTTCCACAATGCAAAGTT-3′

Figure 1

Mass spectroscopic data of isolated chelidonine and the chemical structure"

Figure 2

Effects of different concentrations of chelidonine on HeLa cells incubated for 48 h tested by MTT assayData were expressed as mean±standard error of mean. Values were measured in three independent experiments done in triplicate. **P<0.01, vs control group. MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide."

Figure 3

Effects of different concentration of chelidonine on normal peripheral blood mononuclear cells incubated for 48 h tested by MTT assayData were expressed as mean±standard error of mean. Values were measured in three independent experiments done in triplicate. *P<0.05, **P<0.01, vs control group. MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide."

"

Group n Apoptotic percentage Necrotic percentage
Control 3 20.278±0.003 3.307±0.003
D1 (22.5 μg/mL) 3 40.435±0.024** 4.467±0.005**
D2 (30.0 μg/mL) 3 44.037±0.009** 6.459±0.007**
D3 (37.5 μg/mL) 3 45.207±0.023** 10.333±0.004**

Figure 4

Morphological changes of HeLa cells for 48 h of treatment (phase-contrast microscopy, ×100) D1: 22.5 μg/mL; D2: 30.0 μg/mL; D3: 37.5 μg/mL. "

Figure 5

Control and drug-treated cells stained with DAPI (fluorescence microscopy, ×200) D1: 22.5 μg/mL; D2: 30.0 μg/mL; D3: 37.5 μg/mL. DAPI: 4′,6-diamidino-2-phenylindole. "

Figure 6

Control and drug-treated cells stained with AO-EB (fluorescence microscopy, ×200) D1: 22.5 μg/mL; D2: 30.0 μg/mL; D3: 37.5 μg/mL. AO-EB: acrydine orange-ethidium bromide. "

Figure 7

DNA damage tested by comet assay (fluorescence microscopy, ×200) D1: 22.5 μg/mL; D2: 30.0 μg/mL; D3: 37.5 μg/mL. "

Figure 8

DNA fragmentation assay of HeLa cells treated for 48 h of treatmentD1: 22.5 μg/mL; D2: 30.0 μg/mL; D3: 37.5 μg/mL. "

Figure 9

Circular dichroism spectra of calf thymus DNA incubated with chelidonine"

Figure 10

FACS analysis by annexin V-FITC/PI assayApoptotic cells (annexin V-FITC/PI) were analyzed by FACS and dot plots display Annexin V fluorescence (X-axis) versus PI fluorescence (Y-axis) with respect to the control cells after 48 h of treatment. D1: 22.5 μg/mL; D2: 30.0 μg/mL; D3: 37.5 μg/mL. FACS: fluorescence-activated cell sorter; FITC: fluorescein isothiocyanate; PI: propidium iodide. "

Figure 11

FACS analysis for cell cycle arrest in HeLa cellsCells were harvested and processed for cell cycle analysis using PI. Cell cycle phase distribution of HeLa cell nuclear DNA was determined by single label by FACS. Histogram display of DNA content (X-axis, PI fluorescence) versus counts (Y-axis) has been shown (sub-G1, G0/G1, S, and G2/M) for 48 h of treatment. D1: 22.5 μg/mL; D2: 30.0 μg/mL; D3: 37.5 μg/mL. FACS: fluorescence-activated cell sorter; PI: propidium iodide. "

Figure 12

ROS generation in HeLa cells observed by fluorescence microscopic observation and measured by FACS analysis =A: Fluorescence microscopic observation of ROS generation in HeLa cells (×200); B: FACS analysis of ROS generation in the D1-, D2-, and D3-treated cells with respect to the control cells. Control and treated cells were incubated with H2DCHF-DA fluorescent probe and analyzed in a single labelling FACS system at 530 nm band pass filter using histogram plot. D1: 22.5 μg/mL; D2: 30.0 μg/mL; D3: 37.5 μg/mL. ROS: reactive oxygen species; FACS: fluorescence-activated cell sorter; PI: propidium iodide; DCHF-DA: 2′,7′-dichlorodihydrofluor-escein diacetate."

Figure 13

FACS analysis of changes in MMP in HeLa cells Control and treated cells were incubated with Rhodamine 123 fluorescent probe and analyzed in a single labelling FACS system at 530 nm band pass filter using histogram plot. D1: 22.5 μg/mL; D2: 30.0 μg/mL; D3: 37.5 μg/mL. FACS: fluorescence-activated cell sorter; MMP: mitochondrial membrane potential."

Figure 14

Western blot analysis of p38, p53, caspase 3, PI3K, E6, E7 and GAPDHThe expressions of p38, p53 and caspase3 were up-regulated in the drug-treated cells with respect to the control cells whereas PI3K expression was down-regulated. GAPDH acted as a house-keeping gene. Ln1: Control; Ln2: 22.5 μg/mL; Ln3: 30.0 μg/mL; Ln4: 37.5 μg/mL. PI3K: phosphatidylinositol 3-kinases; GAPDH: glyceraldehyde-3-phosphate dehydrogenase."

Figure 15

Up-regulated proteins and down-regulated proteins measured by ELISAA: Up-regulated protein expression. B: Down-regulated protein expression."

Figure 16

RT-PCR analysis of p53, Bax, caspase 3, PARP-1, PI3K, AKT, Bcl-2, ICAD and GAPDHThe expressions of p53, Bax, caspase 3 and PARP-1 were up-regulated in drug-treated cells with respect to the positive control while the expressions of PI3K, AKT, Bcl-2 and ICAD were down-regulated. GAPDH acted as a housekeeping gene. RT-PCR: reverse transcription-polymerase chain reaction; PARP-1: poly adenosine diphosphate ribose polymerase-1; PI3K: phosphatidyl inositol 3-kinase; AKT: protein kinase B; Bax: Bcl-2-associated X protein; Bcl-2: B-cell lymphoma 2; ICAD: inhibitor of caspase-activated DNase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase."

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