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. 2013 Oct 20:13:271.
doi: 10.1186/1472-6882-13-271.

Inhibition of Raf-MEK-ERK and hypoxia pathways by Phyllanthus prevents metastasis in human lung (A549) cancer cell line

Sau Har Lee, Indu Bala Jaganath, Rishya Manikam, Shamala Devi Sekaran  1
Affiliations

Inhibition of Raf-MEK-ERK and hypoxia pathways by Phyllanthus prevents metastasis in human lung (A549) cancer cell line

Sau Har Lee et al. BMC Complement Altern Med. .

Abstract

Background: Lung cancer constitutes one of the malignancies with the greatest incidence and mortality rates with 1.6 million new cases and 1.4 million deaths each year. Prognosis remains poor due to deleterious development of multidrug resistance resulting in less than 15% lung cancer patients reaching five years survival. We have previously shown that Phyllanthus induced apoptosis in conjunction with its antimetastastic action. In the current study, we aimed to determine the signaling pathways utilized by Phyllanthus to exert its antimetastatic activities.

Methods: Cancer 10-pathway reporter array was performed to screen the pathways affected by Phyllanthus in lung carcinoma cell line (A549) to exert its antimetastatic effects. Results from this array were then confirmed with western blotting, cell cycle analysis, zymography technique, and cell based ELISA assay for human total iNOS. Two-dimensional gel electrophoresis was subsequently carried out to study the differential protein expressions in A549 after treatment with Phyllanthus.

Results: Phyllanthus was observed to cause antimetastatic activities by inhibiting ERK1/2 pathway via suppression of Raf protein. Inhibition of this pathway resulted in the suppression of MMP2, MMP7, and MMP9 expression to stop A549 metastasis. Phyllanthus also inhibits hypoxia pathway via inhibition of HIF-1α that led to reduced VEGF and iNOS expressions. Proteomic analysis revealed a number of proteins downregulated by Phyllanthus that were involved in metastatic processes, including invasion and mobility proteins (cytoskeletal proteins), transcriptional proteins (proliferating cell nuclear antigen; zinc finger protein), antiapoptotic protein (Bcl2) and various glycolytic enzymes. Among the four Phyllanthus species tested, P. urinaria showed the greatest antimetastatic activity.

Conclusions: Phyllanthus inhibits A549 metastasis by suppressing ERK1/2 and hypoxia pathways that led to suppression of various critical proteins for A549 invasion and migration.

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Figures

Figure 1
Figure 1
Expression level of ten cellular signaling pathways in A549 cells treated with (a) aqueous Phyllanthus extracts and (b) methanolic Phyllanthus extracts. Error bar indicates the standard error of the mean of three independent experiments. PN – P. niruri, PU – P. urinaria, PW – P. watsonii, PA – P. amarus. *P < 0.05 vs untreated-control.
Figure 2
Figure 2
Percentage of cell cycle phase distribution of A549 cells treated with both aqueous and methanolic Phyllanthus extracts and standard drugs at their IC50 (μg/ml) concentrations for (a) 24 hours, (b) 48 hours, and (c) 72 hours. Error bar indicates the standard error of the mean of three independent experiments. Aq – Aqueous, MeOH - Methanolic, Control – Untreated cells. *P < 0.05 vs untreated-control.
Figure 3
Figure 3
Expression level of Pan-Ras, c-Raf, c-Jun/AP-1, Elk-1, c-Myc, HIF-1α, Bcl-2, and FUSE-binding proteins in (a) untreated A549 cells and cells treated with (b) aqueous P. niruri, (c) aqueous P. urinaria, (d) aqueous P. watsonii, (e) aqueous P. amarus, (f) methanolic P. niruri, (g) methanolic P. urinaria, (h) methanolic P. watsonii, and (i) methanolic P. amarus , (j) Expression level of VEGF in untreated-control and Phyllanthus -treated A549 cells, (k) Percentage of individual protein expression analysed using Image J software.
Figure 4
Figure 4
Matrix metalloproteinases (MMPs) expression level in A549 cells treated with (a and c) aqueous Phyllanthus extracts and (b and d) methanolic Phyllanthus extracts. M – protein marker, C – untreated control, L – treatment at 200 μg/ml and 20 μg/ml for aqueous and methanolic extracts respectively, I – treatment at their respective IC50 concentrations, H – treatment at 500 μg/ml and 50 μg/ml for aqueous and methanolic extracts respectively, PN – P. niruri, PU – P. urinaria, PW – P. watsonii, PA – P. amarus.
Figure 5
Figure 5
Expression level of (a) iNOS and (b) VEGF in untreated and Phyllanthus-treated A549 cells. APN - aqueous P. niruri, APU - aqueous P. urinaria, APW - aqueous P. watsonii, APA - aqueous P. amarus, MPN - methanolic P. niruri, MPU - methanolic P. urinaria, MPW - methanolic P. watsonii, MPA - methanolic P. amarus, CIS – Cisplatin, DOX – Doxorubicin. Error bar indicates the standard error of the mean of three independent experiments. P < 0.05 for all extracts-treated A549 compared to untreated-control A549.
Figure 6
Figure 6
Representative 2D-PAGE gels for (a and b) untreated-control A549 and its aqueous P. watsonii -treated A549, as well as (c and d) untreated-control A549 and its methanolic P. watsonii -treated A549.
Figure 7
Figure 7
Clusters of Orthologous Groups (COGs) classification of identified proteins in A549 cells treated with (a) aqueous Phyllanthus extracts and (b) methanolic Phyllanthus extracts.
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