Analyzing Cytotoxic and Apoptogenic Properties of Scutellaria litwinowii Root Extract on Cancer Cell Lines

Abstract

The Scutellaria species (Lamiaceae) is used as a source of flavonoids to treat a variety of diseases in traditional medicine. In spite of many reports about the cytotoxic and antitumor effects of some species of this genus, anticancer researches on one of the Iranian species S. litwinowii have not yet been conducted. The cytotoxic properties of total methanol extract of S. litwinowii and its fractions were investigated on different cancer cell lines including AGS, HeLa, MCF-7, PC12 and NIH 3T3. Meanwhile, the role of apoptosis in this toxicity was explored. The cells were cultured in DMEM medium and incubated with different concentrations of herb plant extracts. Cell viability was quantitated by MTT assay. Apoptotic cells were determined using propidium iodide staining of DNA fragmentation by flow cytometry (sub-G1 peak). Scutellaria litwinowii inhibited the growth of malignant cells in a dose-dependent manner. Among solvent fractions of S. litwinowii, the methylene chloride fraction was found to be more toxic compared to other fractions. The IC(50) values of this fraction against AGS, HeLa, MCF-7 and PC12 cell lines after 24 h were determined, 121.2 ± 3.1, 40.9 ± 2.5, 115.9 ± 3.5 and 64.5 ± 3.4 μg/ml, respectively. Scutellaria litwinowii induced a sub-G1 peak in the flow cytometry histogram of treated cells compared to control cells indicating that apoptotic cell death is involved in S. litwinowii toxicity. Scutellaria litwinowii exerts cytotoxic and proapototic effects in a variety of malignant cell lines and could be considered as a potential chemotherapeutic agent in cancer treatment.

Figure 1

Partitioning scheme using immiscible solvents.

Figure 2

Dose-dependent growth inhibition of malignant cell lines by total methanol extract (5–1000 μg ml⁻¹) after 48 h. Viability was quantitated by MTT assay. The toxicity started at a concentration as little as 80 μg ml⁻¹ and the dose inducing IC₅₀ against AGS, HeLa, MCF-7 and PC12 was calculated 216.1, 250.0, 723 and 243.9, respectively. Results are mean ± SD (n = 3).**P < .01 and ***P < .001 compared to control.

Figure 3

Dose-dependent growth inhibition of malignant cell lines by defatted fraction (6.25–1280 μg ml⁻¹) after 24 h. Viability was quantitated by MTT assay. The IC₅₀ of defatted extract against AGS, HeLa, MCF-7 and PC12 was 372.0, 888.1, 1150 and 567.5 for 24 h, respectively. Results are mean ± SD (n = 3). ***P < .001 compared to control.

Figure 4

Dose-dependent growth inhibition of malignant cell lines by CH₂Cl₂ fraction (5–160 μg ml⁻¹) after 24 h. Viability was quantitated by MTT assay. The IC₅₀ values of this fraction against AGS, HeLa, MCF-7 and PC12 cell lines after 24 h were determined, 121.2 ± 3.1, 40.9 ± 2.5, 115.9 ± 3.5 and 64.5 ± 3.4 μg ml⁻¹, respectively. Results are mean ± SD (n = 3). *P < .05, **P < .01 and ***P < .001 compared to control.

Figure 5

Growth inhibition of HeLa cells by CH₂Cl₂ fraction (50 and 250 μg ml⁻¹) compare with baicalein (50 and 100 μm) after 24 h. Viability was quantitated by MTT assay. CH₂Cl₂ fraction could inhibit the proliferation of cells that is comparable with baicalein. Results are mean ± SD (n = 3). ***P < .001 compared to control.

Figure 6

Flow cytometry histograms of apoptosis assays by PI method in HeLa cells. Cells were treated with 50 μg ml⁻¹ of CH₂Cl₂ fraction for 24 h. Sub-G1 peak as an indicative of apoptotic cells, was induced in CH₂Cl₂ fraction treated but not in control cells. (1) control, (2) serum free (positive control); and (3) CH₂Cl₂ fraction. Flow cytometry histogram of positive control in which cells were cultured in serum free medium and CH₂Cl₂ fraction-treated cells. CH₂Cl₂ fraction-treated cells exhibited a sub-G1 peak in HeLa cells that indicates the involvement of an apoptotic process in CH₂Cl₂ fraction-induced cell death.

Figure 7

Approach to analyzing cytotoxic and apoptogenic properties of S. litwinowii root extract on cancer cell lines.