Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014;2014:260210.
doi: 10.1155/2014/260210. Epub 2014 Jul 22.

Effects of crude extracts from medicinal herbs Rhazya stricta and Zingiber officinale on growth and proliferation of human brain cancer cell line in vitro

Ayman I Elkady  1 , Rania Abd El Hamid Hussein  2 , Osama A Abu-Zinadah  3
Affiliations
Free PMC article

Effects of crude extracts from medicinal herbs Rhazya stricta and Zingiber officinale on growth and proliferation of human brain cancer cell line in vitro

Ayman I Elkady et al. Biomed Res Int. 2014.
Free PMC article

Abstract

Hitherto, limited clinical impact has been achieved in the treatment of glioblastoma (GBMs). Although phytochemicals found in medicinal herbs can provide mankind with new therapeutic remedies, single agent intervention has failed to bring the expected outcome in clinical trials. Therefore, combinations of several agents at once are gaining increasing attractiveness. In the present study, we investigated the effects of crude alkaloid (CAERS) and flavonoid (CFEZO) extracts prepared from medicinal herbs, Rhazya stricta and Zingiber officinale, respectively, on the growth of human GBM cell line, U251. R. stricta and Z. officinale are traditionally used in folkloric medicine and have antioxidant, anticarcinogenic, and free radical scavenging properties. Combination of CAERS and CFEZO treatments synergistically suppressed proliferation and colony formation and effectively induced morphological and biochemical features of apoptosis in U251 cells. Apoptosis induction was mediated by release of mitochondrial cytochrome c, increased Bax : Bcl-2 ratio, enhanced activities of caspase-3 and -9, and PARP-1 cleavage. CAERS and CFEZO treatments decreased expression levels of nuclear NF-κBp65, survivin, XIAP, and cyclin D1 and increased expression level of p53, p21, and Noxa. These results suggest that combination of CAERS and CFEZO provides a useful foundation for studying and developing novel chemotherapeutic agents for the treatment of GBM.

Figures

Figure 1
Figure 1
Combination of CAERS and CFEZO acted synergistically to inhibit cell proliferation and colony formation in U251 cells. The U251 cells were seeded, at a density of 104/well in 96-well plates and treated with the indicated concentrations of CAERS and/or CFEZO for displayed time intervals. The inhibition of cell proliferation was assessed by the tetrazolium salt WST-1 kit as detailed in Section 2. The experiments were repeated five times in triplicate, and cell viabilities at each dose of extract(s) were expressed in terms of percent of control and reported as the mean ± SD. (b) Left histogram: multiple comparisons for effect of different doses' categories on U251 cells using post hoc test (Dunnett T3). All reported P values are 2-tailed; ∗∗correlation is significant at the 0.01 level (2-tailed); ∗∗∗correlation is significant at the 0.001 level (2-tailed); a = significant differences compared to control; b = significant differences compared to the lowest dose, 10 μg/mL. Compared to control, doses in 10, 20, 40, and 50 μg/mL showed a significantly lower percentage of viable U251 cells (P = 0.002∗∗, P = 0.000∗∗∗, P = 0.000∗∗∗, and P = 0.000∗∗∗, resp.). We observed another significant variation between dose 10 μg/mL and doses 40 and 50 μg/mL, with a stronger suppression of GBM cells with the two higher doses (40 and 50 μg/mL) (P = 0.008∗∗, and P = 0.001∗∗∗, resp.). Right histogram: multiple comparisons for effect of different durations on U251 viable cells using post hoc test (LSD). All reported P values are 2-tailed. ∗Correlation is significant at the 0.05 level (2-tailed). Compared to one-day duration, three-day duration of treatment showed a significantly lower percentage of viable U251 cells (P = 0.02∗). The table (on the right) shows regression of dose and duration with percent U251 viable cells. A multiple linear regression analysis was used (Enter Selection Procedure).∗∗∗Correlation is significant at 0.001 level (2-tailed). Dose had a stronger negative impact than that of duration, in predicting the % U251 viable cells, (Beta = −0.84∗∗∗ and Beta = −0.34∗∗∗ resp.). The R 2 indicates that 81.3% of the variation in % U251 viable cells could be explained by these two variables (dose and duration). (c) The MCF-7, HeLa, and HF-5 cell lines were seeded and treated with the indicated concentrations of CAERS and/or CFEZO for displayed time intervals. The experiments were repeated five times in triplicate, and cell viabilities at each dose of extract(s) were expressed in terms of percent of control and reported as the mean ± SD. (d) U251 cells were seeded onto a 6-well plate at 1000 cells/well and treated with the indicated concentrations of CAERS and/or CFEZO as detailed in Section 2. The colonies were counted under a dissection microscope and the experiment was repeated three times. (e) CAERS and/or CFEZO acted synergistically to inhibit anchorage-independent growth in U251 cells in growth in soft agarose assays. U251 cells were plated, in triplicate, in 0.35% soft agarose and treated with CAERS (25 μg/mL) or CFEZO (25 μg/mL) and a combination of CAERS (5 μg/mL) and CFEZO (5 μg/mL). After 2 weeks, the colonies were stained with 0.0005% crystal violet and photographed using a digital camera coupled to a Carl Zeiss inverted microscope. Representative images of colonies in soft agar are shown.
Figure 2
Figure 2
CAERS and CFEZO treatments induced apoptotic cell death. (a) Flow cytometric analyses of apoptosis and necrosis using Annexin V-FITC/PI staining. Panels I, II, III, and IV represent cells treated with vehicle, CAERS (100 μg/mL), CFEZO (100 μg/mL), and combined treatment of CAERS (20 μg/mL) and CFEZO (20 μg/mL), respectively. Cells in left lower quadrants represent viable population (annexin V-negative and PI-negative); cells in right lower quadrants represent early apoptotic population (annexin V-positive, PI-negative); cells in right top quadrants represent late apoptotic population (annexin V-positive and PI-positive) and cells in left top quadrants represent necrotic population (annexin V-negative and PI-positive). (b) Microphotographs showing CAERS and CFEZO treatments induced morphological features of apoptosis in U251 cells. The cells were treated with the indicated concentrations of CAERS and/or CFEZO for 48 h. Then, the photographs were taken directly from culture plates using a phase contrast microscope. Magnification of micrographs was as follows: I: 20x; II: 40x; and III: 63x. (c) Toluidine blue-stained semithin sections. The cells were treated and stained with toluidine blue, as detailed in Section 2. Depicted results are representative for independent experiments with almost identical observations.
Figure 3
Figure 3
Combination of CAERS and CFEZO induced an early biochemical feature of apoptosis. U251 cells were treated with indicated concentrations of CAERS and for 24 h and assayed for existence of apoptotic cell death. Depicted results are representative for independent experiments with almost identical observations. (a) DAPI staining showing combination of CAERS and CFEZO induced nuclear condensation, DNA fragmentation, and perinuclear apoptotic bodies in U251 cells (arrows). (b) Agarose gel showing CAERS and CFEZO induced DNA fragmentation in U251 cells. Lane “M” indicates the DNA marker ladder. (c) Comet assay showing formation of DNA tail in CAERS- and CFEZO-treated U251 cells. Nuclei with damaged DNA have the appearance of a Comet with a bright head and a tail, whereas nuclei with undamaged DNA appear round with no tail. In the panel denoted with 24 h, cells were treated with 20 μg CAERS and 20 μg CFEZO for 24 h; then treatment medium was discarded, cells were washed and grown in CAERS- and CFEZO-free medium for 24 before being harvested and assayed for comet analysis. The histogram displays percentage of cells with comet tail being analyzed in 50 cells for one slide. The bar denoted with 24, at the top, represents cells treated for 24 h, which then were grown in CAERS- and CFEZO-free medium for 24.
Figure 4
Figure 4
CAERS and CFEZO treatments trigger mitochondrial-dependent apoptotic pathway. U251 cells were treated with indicated concentrations of CAERS, CFEZO, and combination of CAERS CFEZO for 24 h and assayed as detailed in Section 2. (a) Immunoblots showing CAERS and/or CFEZO treatments mediated mitochondrial cyto c release, activation of caspases 9 and 3, and PARP-1 cleavage. The mitochondrial marker, anticytochrome oxidase IV (COXIV), shows the purity of the cytoplasmic fraction and equal loading of the mitochondrial fraction. (b) CAERS and/or CFEZO treatment(s) altered expression ratio of Bax/Bcl-2 at protein and mRNA levels, in favor of apoptosis. The histogram depicts the Bcl-2 (dark bars) and Bax (light bars) mRNA ratio measured by using densitometric analysis. In all western blot analyses, the membranes were stripped and reprobed with antiactin antibody as a loading control.
Figure 5
Figure 5
(a) CAERS and/or CFEZO treatment(s) modulated the expression of the NF-κBp65 and p53 proteins. The U251 cells (20 × 104 cells/well) were seeded onto 6-well plates and treated with the indicated concentrations of CAERS and/or CFEZO for 24 h. Subsequently, 20 μg of whole cell (or nuclear) protein extract was isolated from treated cells and subjected to SDS-PAGE in 10% polyacrylamide gels, transferred to PVDF membranes, and immunoblotted with antibodies against the depicted proteins. The immunoblots showed that CAERS and/or CFEZO treatment(s) downregulated expression level of NF-κBp65 protein found in whole cell or nuclear extract but did not alter level of NF-κBp65 found in the cytosolic extract. The immunoblot showing nuclear NF-κBp65 was stripped off and reprobed with anti-PCNA antibody as an internal loading control for nuclear extract. The CAERS and/or CFEZO treatment(s) upregulated expression level of p53 protein. Representative blots from several independent experiments are shown. (b) CAERS and/or CFEZO treatment(s) modulated the expression of the displayed antiapoptotic and proapoptotic gene products. After CAERS and/or CFEZO treatment(s) total RNA was then isolated, reverse-transcribed, and subjected to PCR with gene-specific primers. The PCR products of the genes were then subjected to electrophoresis in 1% agarose gels and visualized by staining with ethidium bromide. GAPDH was used as the internal control, M, DNA ladder. The data are representative of three separate experiments.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by 7 articles

See all "Cited by" articles

References

    1. Preusser M, de Ribaupierre S, Wöhrer A, et al. Current concepts and management of glioblastoma. Annals of Neurology. 2011;70(1):9–21. - PubMed
    1. Krakstad C, Chekenya M. Survival signalling and apoptosis resistance in glioblastomas: opportunities for targeted therapeutics. Molecular Cancer. 2010;9, article 135 - PMC - PubMed
    1. Cui Y, Wang Q, Wang J, et al. Knockdown of AKT2 expression by RNA interference inhibits proliferation, enhances apoptosis, and increases chemosensitivity to the anticancer drug VM-26 in U87 glioma cells. Brain Research. 2012;1469:1–9. - PubMed
    1. Wong RSY. Apoptosis in cancer: from pathogenesis to treatment. Journal of Experimental and Clinical Cancer Research. 2011;30(1, article 87) - PMC - PubMed
    1. Earnshaw WC, Martins LM, Kaufmann SH. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annual Review of Biochemistry. 1999;68:383–424. - PubMed

Publication types

MeSH terms