Mycochemistry, antioxidant content, and antioxidant potentiality of the ethanolic extract of Pleurotus florida and its anti-cancerous effect on HeLa cancer cell line, and antitumor effect on HeLa-implanted mice

Abstract

Objectives: Cervical cancer is increasing worldwide and is becoming resistant to the existing drugs in clinical practice. Here, ethanolic extract of fruit body of Pleurotus florida was evaluated as antioxidant, anticancer agent against HeLa cell lines and anti-tumor against cervical cancer in mice model.

Methods: Fruit bodies of P. florida in 90% ethanol, and the P. florida ethanolic extract (PFEE) was subsequently investigated for its antioxidant content and activity, anticancer properties against the cervical cancer cell line, HeLa, and antitumor activity against HeLa implanted mice.

Results: The antioxidant activity bioassay showed that the IC₅₀ of PFEE was 41.17 ± 1.42a μg/ml. The cytotoxicity assay revealed that PFEE caused inhibition of cell proliferation. At the highest dose (1,250 μg/ml) after 24 h, 48 h, or 72 h of treatment, the percentages of cell growth inhibition were 75.22%, 77.77%, and 84.65%, respectively. It revealed that PFEE-treated cells became rounded and the nuclei became fragmented. PFEE induced intracellular generation of reactive oxygen species and reduced the mitochondrial membrane potential. PFEE also led to an up regulation of the apoptotic genes for caspases-3, -9, and Bax, whereas Bcl-2 gene was down regulated, and it also promoted the expression of p53. Cell cycle analysis revealed that cell cycle was arrested at the G₀/G₁ checkpoint. PFEE suppressed metastasis and colonization. At a dosage of PFEE of 50 mg/kg of body weight, a 66.72% reduction in the size of tumors and an 87.44% reduction in the tumor weight were observed in mice.

Conclusions: It has demonstrated that PFEE is a highly potent anti-cervical cancer agent in vitro and in vivo.

Keywords: Apoptosis; Cytotoxicity; colonization; metastasis; migration.

Figure 1

Effect of PFEE on HeLa cell morphology under phase contrast microscope(20×). (a) Control cells showed normal spindle shaped morphology but (b-d) treated cell showed mostly round shaped morphology after 24 h treatment of PFEE

Figure 2

Effect of PFEE on HeLa nuclear morphology under Inverted Fluorescence microscopy. (a) Control showed Hoechst-33342 stained round nucleus of HeLa cell. (b and c) showed Hoechst-33342 stained nucleus of PFEE (500 and 1000 μg/ml) treated HeLa cell and they were condensed and fragmented. “Arrow” indicates nuclear fragmentations

Figure 3

Cytotoxic effect of PFEE on HeLa cell at (a) 24 h, (b) 48 h, (c) 72 h. in vitro cytotoxic effect of PFEE (0–1250 μg/ml was measured by MTT assay on HeLa cell line. Bar graphs shows % of viable cells (Y axis) against PFEE concentration (X axis). (a-c) demonstrates decreasing percentage of viable cells with increasing concentration of PFEE as compared to untreated cell. Data are representative of three independent experiment and bar graph showed mean ±SEM (**P < 0.05, ***P < 0.001, ns not significant)

Figure 4

Flow cytometric detection of the effect of PFEE on HeLa cell cycle phase distribution. (a) Control. (b) 1000 μg/ml. (c) 1250 μg/ml (d) 1500 μg/ml shows interfere with cell cycle population by inducing G₀/G₁ arrest of HeLa cell in vitro, in 24 h

Figure 5

Demonstrates intracellular ROS generation by PFEE in HeLa cell. Treated cells showed high-intensity green fluorescent as compared to control cell, here ascorbic acid and H₂O₂ used as a negative and positive control

Figure 6

Fluorescence microscopic (10×) images of HeLa cells after JC1 staining. Control cell showed no MMP change as they are mostly red (JC1 aggregate), but treated cells are mostly green (JC1 monomeric)

Figure 7

Agarose gel (1.5%) electrophoresis of isolated HeLa cell DNA for 24,48 and 72 h. Control (c) lane showed no fragmented DNA but 1000 μg/ml showed heavily fragmented DNA in the form of ladder after 72 h.

Figure 8

Effect of PFEE on migration of HeLa Cells in scratch method. (a) At 0 h no cell migration in control and treated (500 and 1000 μg/ml) and after 24 h control showed scratch/distance is almost filled by migrated cells but treated showed some cells are migrated in the scratch. (b) Showed bar graph of percentage of cell migrated (Y axis) against concentration of PFEE (X axis). Data are representative of three independent experiments and bar graph shows mean ± SEM (**P < 0.05, ***P < 0.001, not significant).

Figure 9

Effect of PFEE on colony formation of HeLa cells in vitro. (a) Showed dose dependent inhibition of clonogenicity in HeLa cell. (b) Showed bar graph of percentage of colonies (Y axis) against concentration of PFEE (X axis). Data are representative of three independent experiments and bar graph shows mean ± SEM (**P < 0.05, ***P < 0.001, ns not significant)

Figure 10

Effects of PFEE on tumor growth. PFEE suppresses the growth of cervical cancer tumors in Swiss albino mice. Mice were inoculated subcutaneously in the right flank with 2 × 10⁶ HeLa cells. Tumor volume was measured twice/week using a caliper and calculated as (width) 2 × length/2. Representative images were captured at the end of therapy, (a) shows measurement of tumor treated (50mg PFEE/Kg) and control (HeLa+ vehicle), (b) sacrificed mouse (dissected), (c) tumor from PFEE (10 mg/kg) treated mouse, (d) tumor from PFEE (50 mg/kg) treated mouse

Figure 11

Graphical representation (line plot) of the effects of PFEE on tumor growth in vivo mg/kg indicates mg PFEE per kg body weight of mice. The data represented as mean ±SD for the three different experiments performed in triplicate. Error bars of ± SD are inserted in figure