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Journal of Chinese Integrative Medicine ›› 2013, Vol. 11 ›› Issue (6): 405-415.doi: 10.3736/jintegrmed2013057

• Research Article • Previous Articles     Next Articles

Diarylheptanoid-myricanone isolated from ethanolic extract of Myrica cerifera shows anticancer effects on HeLa and PC3 cell lines: Signalling pathway and drug-DNA interaction

Paul Avijita, Das Sreemantia, Das Jayeetaa, Samadder Asmitaa, Bishayee Kausika, Sadhukhan Ratanb, Anisur Rahman Khuda-Bukhsha()   

  1. a. Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani,Kalyani-741235, India
    b. Department of Biochemistry and Biophysics, University of Kalyani, Kalyani-741235, India
  • Received:2013-06-11 Accepted:2013-08-01 Online:2013-11-10 Published:2013-11-15

Objective

To test if myricanone (C21H24O5), a cyclic diarylheptanoid, has anticancer effects on two different cancer cell lines HeLa and PC3. The present study was conducted with a note on the drug-DNA interaction and apoptotic signalling pathway.


Methods

Several studies like cytotoxicity, nuclear damage, annexin-V-fluorescein isothiocyanate (FITC)/propidium iodide (PI)-labelled apoptotic assay and cell cycle arrest, immunoblot and reverse transcriptase-polymerase chain reaction (RT-PCR) were used following standard protocols. Circular dichroism (CD) spectroscopy was also done to evaluate whether myricanone effectively interacted with DNA to bring about conformational changes that could strongly inhibit the cancer cell proliferation.


Results

Myricanone showed a greater cytotoxic effect on PC3 cells than on HeLa cells. Myricanone promoted G0/G1 arrest in HeLa cells and S phase arrest in PC3 cells. Nuclear condensation and annexin V-FITC/PI studies revealed that myricanone promoted apoptotic cell death. CD spectroscopic data indicated that myricanone had an interaction with calf thymus DNA that changed DNA structural conformation. RT-PCR and immunoblot studies revealed that myricanone activated the apoptotic signalling cascades through down-regulation of transcription factors like nuclear factor-κB (NF-κB) (p65), and signal transducers and activators of transcription 3 (STAT3); cell cycle regulators like cyclin D1, and survivin and other signal proteins like Bcl-2 and up-regulation of Bax, caspase-9 and caspase-3.


Conclusion

Myricanone induced apoptosis in both types of cancer cells by triggering caspase activation, and suppression of cell proliferation by down-regulation of NF-κB and STAT3 signalling cascades, which makes it a suitable candidate for possible use in the formulation of therapeutic agent for combating cancer.

Key words: Myrica, DiarylHeptanoids, Plant extracts, Apoptosis, NF-κB;, STAT3 transcription factor, Signal transduction

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Figure 1

Mass spectroscopic data and chemical structure of myricanone"

Figure 2

Analysis of cytotoxic and apoptotic potentials of myricanoneCells were cultured in presence of various concentrations of myricanone (10-50 μg/mL) for 48 h. Cell proliferation was measured by MTT assay. IC50 value of myricanone on HeLa and PC3 cells were found to be 29.6 μg/mL and 18.4 μg/mL, respectively. Data are expressed as mean ± standard error of mean, n=3.HaCaT: immortal human keratinocyte line; PBMC: peripheral blood mononuclear cell; HeLa: human cervical cancer cell line; PC3: human prostate cancer cell line; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; IC50: half inhibitory concentration."

Figure 3

Morphological changes of control and D1-, D2-, and D3-treated HeLa and PC3 cells for 48 h of myricanone treatment (Phase contrast microscopy, ×200) Myricanone showed greater apoptotic effect on both HeLa and PC3 cells in a dose-dependent manner. In higher doses, more apoptotic round-shaped, cellular blebbing, and shrinked cells were observed in both cells.D1: 10 μg/mL; D2: 20 μg/mL; D3: 30 μg/mL."

Figure 4

Analysis of drug-DNA interaction and DNA damage induced by myricanone CD spectra of calf thymus DNA were incubated with myricanone.CT-DNA: calf thymus DNA; CD: circular dichroism."

Figure 5

Nuclear condensation assessment of control and drug-treated cells by DAPI staining (Fluorescence microscopy, ×200) After drug treatment the numbers of apoptotic nuclei were increased and more nuclear condensation was observed in both cells with respect to control cells. Arrows showed the apoptotic nuclei.DAPI: 4',6-diamidino-2-phenylindole. D1: 10 μg/mL; D2: 20 μg/mL; D3: 30 μg/mL."

Figure 6

DNA fragmentation assay of HeLa and PC3 cells treated with control and D1, D2, and D3. D1: 10 μg/mL; D2: 20 μg/mL; D3: 30 μg/mL."

Figure 7

Flow cytometric analysis of myricanone-mediated apoptosis in HeLa and PC3 cells by annexin V-FITC/PI double staining Differentiation of cell population was done on flow cytometer and was assessed as: viable cancer cells (annexin -ve; PI -ve); early apoptotic cancer cells (annexin +ve; PI -ve); late apoptotic cancer cells (annexin +ve; PI +ve); necrotic cells (PI +ve). Percentages of cell population were written in their respective quadrants. Samples were analyzed in Cyflogic (v.1.2.1) software and determination was based on mean fluorescence intensity of 10 000 events. Bar charts show the mean values of unaffected cells, early apoptotic cells, late apoptotic cells and necrotic cells.FITC: fluorescein isothiocyanate; PI: propidium iodide; D1: 10 μg/mL; D2: 20 μg/mL; D3: 30 μg/mL."

Figure 8

Flow cytometric analysis of the cell cycle in myricanone-treated HeLa and PC3 cells with respect to untreated cells In HeLa cells, arrest occurred at G0/G1 stage whereas in PC3 cells arrest occurred at S phase.D1: 10 μg/mL; D2: 20 μg/mL; D3: 30 μg/mL."

Figure 9

Effects of myricanone on mRNA and protein level expressions in HeLa and PC3 cells with respect to untreated cells A: RT-PCR analysis of Bax, Bcl-2 and caspase-3 of control and treated cells. mRNA expression analysis showed up-regulation of Bax, caspase-3 and down-regulation of Bcl-2 activity. B: Expression of Bax, Bcl-2, caspase-9 and caspase-3 of control and treated cells was analyzed by Western blot analysis. Significant up-regulation of Bax, caspase-3, and caspase-9 and down-regulation of Bcl-2 was found in myricanone-treated cells in a dose-dependent manner. The cleavage of caspase-3, which is the active form of caspase-3 was also observed in both cell lines. GAPDH acts as a housekeeping gene. The intensity of the control was normalized to 1, and the intensity of each band from treated cells is compared with the control. RT-PCR: reverse transcriptase-polymerase chain reaction; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; D1: 10 μg/mL; D2: 20 μg/mL; D3: 30 μg/mL."

Figure 10

mRNA and protein level expressions in myricanone-treated HeLa and PC3 cells with respect to untreated cells A: RT-PCR analysis of IL-6, STAT3 and NF-κB. Expression analysis revealed significant down-regulation of IL-6, STAT3 and NF-κB expression when treated with myricanone with respect to control cells. B: Expression of STAT3, phosphorylated STAT3 (Tyrosine 705) and NF-κB (p65) of control and treated cells was analyzed by Western blotting. GAPDH acts as a housekeeping gene. STAT3 and NF-κB expression was found to be significantly down-regulated in myricanone-treated cells compared to untreated control cells. The intensity of the control was normalized to 1, and the intensity of each band from treated cells is compared with the control.NF-κB: nuclear factor-κB; STAT3: signal transducer and activator of transcription 3; IL-6: interleukin-6; RT-PCR: reverse transcriptase-polymerase chain reaction; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; D1: 10 μg/mL; D2: 20 μg/mL; D3: 30 μg/mL."

Figure 11

RT-PCR analysis of cyclin D1 and survivin of control and treated cellsMyricanone-treated cells showed significant down-regulation of cyclin D1 and survivin expression when compared with untreated control cells. GAPDH acts as a housekeeping gene. The intensity of the control was normalized to 1, and the intensity of each band from treated cells is compared with the control.RT-PCR: reverse transcriptase-polymerase chain reaction; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; D1: 10 μg/mL; D2: 20 μg/mL; D3: 30 μg/mL."

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