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Crude Aqueous Extracts of Pluchea Indica (L.) Less. Inhibit Proliferation and Migration of Cancer Cells Through Induction of p53-dependent Cell Death

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Crude Aqueous Extracts of Pluchea Indica (L.) Less. Inhibit Proliferation and Migration of Cancer Cells Through Induction of p53-dependent Cell Death

Jonathan J Cho et al. BMC Complement Altern Med.

Abstract

Background: Pluchea indica (L.) Less. (Asteraceae) is a perennial shrub plant with anti-inflammatory and antioxidant medicinal properties. However, the anti-cancer properties of its aqueous extracts have not been studied. The aim of this study was to investigate the anti-proliferation, anti-migration, and pro-apoptotic properties of crude aqueous extracts of P. indica leaf and root on human malignant glioma cancer cells and human cervical cancer cells, and the underlying molecular mechanism.

Methods: GBM8401 human glioma cells and HeLa cervical carcinoma cells were treated with various concentrations of crude aqueous extracts of P. indica leaf and root and cancer cell proliferation and viability were measured by cell growth curves, trypan blue exclusions, and the tetrazolium reduction assay. Effects of the crude aqueous extracts on focus formation, migration, and apoptosis of cancer cells were studied as well. The molecular mechanism that contributed to the anti-cancer activities of crude aqueous extracts of P. indica root was also examined using Western blotting analysis.

Results: Crude aqueous extracts of P. indica leaf and root suppressed proliferation, viability, and migration of GBM8401 and HeLa cells. Treatment with crude aqueous extracts of P. indica leaf and root for 48 hours resulted in a significant 75% and 70% inhibition on proliferation and viability of GBM8401 and HeLa cancer cells, respectively. Crude aqueous extracts of P. indica root inhibited focus formation and promoted apoptosis of HeLa cells. It was found that phosphorylated-p53 and p21 were induced in GBM8401 and HeLa cells treated with crude aqueous extracts of P. indica root. Expression of phosphorylated-AKT was decreased in HeLa cells treated with crude aqueous extracts of P. indica root.

Conclusion: The in vitro anti-cancer effects of crude aqueous extracts of P. indica leaf and root indicate that it has sufficient potential to warrant further examination and development as a new anti-cancer agent.

Figures

Figure 1
Figure 1
Pro-oxidant activity of crude aqueous extract of P. indica root in HeLa cancer cells. Measurement of thiobarbituric acid reactive species (TBARS) during malondialdehyde peroxidation in HeLa cells treated with 0.5 mg/ml of crude aqueous extracts at 24-hr and 48-hr incubation respectively were performed as described in Methods. Each experiment was done in triplicate. The pro-oxidant activity of the crude aqueous extract of P. indica root was evident. Data are expressed as mean ± SD. *P < 0.01.
Figure 2
Figure 2
Effect of P. indica leaf or root crude aqueous extract on the growth of cancer cells. Growth curves of (A) GBM8401 cells and (B) HeLa cells after 300 μg/ml P. indica leaf or root aqueous extract treatment. Each experiment was done in triplicate. Data are expressed as mean ± SD. *P < 0.05.
Figure 3
Figure 3
Effect of P. indica leaf or root crude aqueous extract on cell proliferation and viability of cancer cells as determined by MTS assay. MTS assay of (A) GBM8401 cells and (B) HeLa cells after 0, 100, 500, and 1000 μg/ml P. indica leaf or root aqueous extract treatment. Each experiment was done in triplicate. Data are expressed as mean ± SD.
Figure 4
Figure 4
Effect of P. indica root crude aqueous extract on the focus formation ability of cancer cells. Focus formation assay of (A, B, C, D) GBM8401 cells and (E, F, G, H) HeLa cells after P. indica root aqueous extract treatment. (A, E) 0 μg/ml P. indica root aqueous extract treatment. (B, F) 10 μg/ml P. indica root aqueous extract treatment. (C, G) 100 μg/ml P. indica root aqueous extract treatment. (D, H) 200 μg/ml P. indica root aqueous extract treatment. Each experiment was repeated five times.
Figure 5
Figure 5
Effect of P. indica root crude aqueous extract on migration of HeLa cells as determined by in vitro “scratch” wounjd closure assay. “Scratch” wound closure assay of (A) HeLa cells after 0 hours. (B) HeLa cells after 48 hours incubation with 0 μg/ml P. indica root aqueous extract. (C) HeLa cells after 48 hours incubation with 250 μg/ml P. indica root aqueous extract. (D) HeLa cells after 48 hours incubation with 500 μg/ml P. indica root aqueous extract. (E) Statistical analysis of HeLa cells after 48 hours incubation with 0, 250, or 500 μg/ml P. indica root aqueous extract. Each experiment was done in triplicate. Error bars show mean ± SD; bars show means; *P < 0.05.
Figure 6
Figure 6
Fluorescence microscopy of HeLa cells treated with crude aqueous root extracts of P. indica for 48 hours and double stained with annexin-V-sensitive probe (conjugated to FITC) and propidium iodide. Qualitative labeling of annexin V in the plasma membrane (B and E) or cellular uptake of propidium iodide (C and F) was recorded. Control cells exposed to PBS (A)-(C), and 0.5 mg/ml of crude aqueous extracts treated cells (D)-(F) were photographed with a microscope (× 40).
Figure 7
Figure 7
Flow cytometry analysis of HeLa cells stained with FITC-Annexin V and PI and Western blotting of enhanced expression of cleaved caspase-3. Flow cytometry analysis of FITC-Annexin V and PI stained HeLa cells with untreated HeLa cells (A) and HeLa cells treated with 0.5 mg/ml P. indica root extract for 48 hours (B). In each scatter plot, upper-left quadrant (Q1-1) shows naked nucleus cell mass, upper-right quadrant (Q2-1) shows necrotic cell mass, lower-left quadrant (Q3-1) shows survival cell mass, and lower-right (Q4-1) shows apoptotic cell mass. In control cells, there were only 0.1% of apoptotic cells (Figure 7A; Q4-1). After 48 hours of treatment with 0.5 mg/ml P. indica root aqueous extract, approximately 43% of cells were detected as undergoing necrosis and apoptosis (Figure 7B; Q2-1+Q4-1). (C) A representative of Western blotting of time-dependent increasing expression of cleaved caspase-3. Cleaved Caspase-3 (Asp175) monoclonal antibody (5A1E; Cell Signaling Technology) used detects levels of the large fragment (17/19 kDa) of activated caspase-3 resulting from cleavage adjacent to Asp175. Lane 1: untreated; lane 2: 1 day of 0.5 mg/ml extract treatment; lane 3: 2 days of 0.5 mg/ml extract treatment; lane 4: 3 days of 0.5 mg/ml extract treatment. Each experiment was done in triplicate.
Figure 8
Figure 8
Western blot analysis of phosphorylated-p53 and p21 proteins in cancer cells treated with P. indica leaf or root aqueous extract. (A) Western blot of p21 and phosphorylated-p53 proteins of GBM8401 cells after P. indica extract treatment. Lane 1: untreated; lane 2: 3 days of 0.1 mg/ml P. indica root aqueous extract treatment; lane 3: 3 days of 1 mg/ml P. indica root aqueous extract treatment; lane 4: 5 days of 1 mg/ml P. indica root aqueous extract treatment; lane 5: 3 days of 0.1 mg/ml P. indica leaf aqueous extract treatment; lane 6: 3 days of 1 mg/ml P. indica leaf aqueous extract treatment. (B) Western blot of p21, phosphorylated-p53, AKT, and phosphorylated-AKT proteins of HeLa cells after P. indica extract treatment. Lane 1: untreated; lane 2: 2 days of 0.25 mg/ml P. indica root aqueous extract treatment; lane 3: 2 days of 0.5 mg/ml P. indica root aqueous extract treatment. Each experiment was done in triplicate.

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