In vitro assessment of anti-proliferative effect induced by alpha-mangostin from Cratoxylum arborescens on HeLa cells

Natural medicinal products possess diverse chemical structures and have been an essential source for drug discovery. Therefore, in this study, α-mangostin (AM) is a plant-derived compound was investigated for the apoptotic effect on human cervical cancer cells (HeLa). The cytotoxic effects of AM on the viability of HeLa and human normal ovarian cell line (SV40) were evaluated by using MTT assay. Results showed that AM inhibited HeLa cells viability at concentration- and time-dependent manner with IC50 value of 24.53 ± 1.48 µM at 24 h. The apoptogenic effects of AM on HeLa were assessed using fluorescence microscopy analysis. The effect of AM on cell proliferation was also studied through clonogenic assay. ROS production evaluation, flow cytometry (cell cycle) analysis, caspases 3/7, 8, and 9 assessment and multiple cytotoxicity assays were conducted to determine the mechanism of cell apoptosis. This was associated with G2/M phase cell cycle arrest and elevation in ROS production. AM induced mitochondrial apoptosis which was confirmed based on the significant increase in the levels of caspases 3/7 and 9 in a dose-dependent manner. Furthermore, the MMP disruption and increased cell permeability, concurrent with cytochrome c release from the mitochondria to the cytosol provided evidence that AM can induce apoptosis via mitochondrial-dependent pathway. AM exerted a remarkable antitumor effect and induced characteristic apoptogenic morphological changes on HeLa cells, which indicates the occurrence of cell death. This study reveals that AM could be a potential antitumor compound on cervical cancer in vitro and can be considered for further cervical cancer preclinical and in vivo testing

Histopathology of the spleen tissues.

<p>(A) Control where BALB/c mice were not injected with WEHI-3 cells; the white pulp, red pulp and lymphocyte cells were clearly shown. (B) BALB/c mice injected with WEHI-3 cells and not treated. The leukemia sectioning showed widened white pulp, but the red pulp became tiny. The yellow arrows indicated neoplastic cells. (C) BALB/c mice injected with WEHI-3 cells and treated with 100 mg/kg thymoquinone. The white pulp decreased in size, and the red pulp exhibited a little increase in size. The treatment with 100 mg/kg thymoquinone showed similarity to the control.</p

Cytotoxicity effect of thymoquinone.

<p>(A) Chemical structure of thymoquinone. (B) The cell viability after 24 h of treatment. Each point is the mean ± S.D. of three independent experiments. (C) Normal phase contrast inverted micrograph for 0, 24, 48, and 72 h. In control, most of the cells exhibited normal morphology while some cells showed cytoplasmic protrusions (24 h); clearly blebbing and apoptosis were observed (48 h); most of the cell exhibited growth inhibition and apoptosis (72 h). White arrows: membrane blebbing; yellow arrows: Apoptosis.</p

Fluorescent microscope analysis of nuclei fragmentation by Hoechst 33342 and AO/PI Double-Staining.

<p>(A) Staining with Hoechst 33342. Cells in the control were untreated WEHI-3 cells. Apoptotic cells appeared (white arrows) after 24 and 48 h. In 72 h, all of the cells were apoptotic. (B) AO/PI staining. After 72 h, the untreated cells showed normal structure without prominent apoptosis and necrosis. Early apoptosis features were seen after 24 h representing intercalated AO (bright green) amongst the fragmented DNA; blebbing, orange color representing late apoptosis were noticed after 48 h treatment; bright red colored secondary necrosis were visible after 72 h. White arrows: viable cells; red arrows: membrane blebbing; pink arrows: early apoptosis; blue arrows: late apoptosis; yellow arrows: secondary necrosis. Images are representative of one of three similar experiments. (C) Early and late apoptosis increased significantly (*p<0.05) compared to control, in a time-dependent manner. However, no significant difference was observed in necrosis cells. VI: viable cell, EA: early apoptosis, LA: late apoptosis, SN: secondary necrosis</p

Detection of apoptosis by TUNEL assay in (A) the spleen and (B) the liver tissues of BALB/c mice.

<p>Normal sections show less apoptotic cells (white arrows). TUNEL staining in positive control section of BALB/c mice (induced with leukemia and without treatment) shows aggressive cell proliferation without apoptotic cells. Tissues of BALB/c mice induced with leukemia and treated with 100 mg/kg thymoquinone (TQ) show the evidence of apoptosis by the represented apoptotic cells (white arrows).</p

Effect of thymoquinone on early apoptosis, cell cycle analysis and protein expression.

<p>(A) Staining with FITC-conjugated Annexin V and PI; cells were analyzed by flow cytometry. Control cells (no drug treatment), 24, 48 and 72 h were in a time-dependent manner. The early apoptotic events (Annexin+/PI-) are shown in lower right quadrant (Q4) of each panel. Quadrant (Q2) represents Annexin+/PI+ late stage of apoptosis/dead cells. (n = 2). (B) Histograms for cell cycle analysis. (C) Cell cycle graph; Induction of S phase arrest in the cell cycle progression. “*” Indicates statistically significant at p<0.05, where the arrest at 24, 48 and 72 h was individually compared to control. (D) Effect of thymoquinone on the levels of apoptosis regulatory proteins at 3, 6, 12 and 24 h with β- actin as a loading control, ‘*’ indicates statistically significant at p<0.05. Shapiro-Wilk test: Hsb70, P = 0.339; Bcl2, P = 0.57; Bax, P = 0.192, where P value are greater than α level of 0.05, showing that the data have normally distributed population.</p

Effects of thymoquinone on the weights of liver and spleen tissues from BALB/c mice.

<p>The animals were injected with WEHI-3 cells (1×10⁶) for 3-week time period and treated with or without thymoquinone (TQ) for 3 weeks. (A) Livers were individually collected, photographed, and (B) weighed. (C) Spleen was individually collected, photographed and (D) weighed. Thymoquinone affected the weight of liver and spleen tissues from BALB/c mice. ‘*’ indicates statistically significant at p<0.05. (n = 5).</p

Effects of thymoquinone on animal body weight.

<p>(A) Representative images of normal BALB/c mice; BALB/c mice injected with WEHI-3 cells (1×10⁶) and BALB/c mice injected with WEHI-3 cells (1×10⁶) and treated with thymoquinone 100 mg/kg for 3 weeks. The animals were then sacrificed, and photographed. The arrows are pointing at the size of the liver (blue arrow) and spleen (red arrow). (B) Body weight changes of BALB/c mice treated with or without thymoquinone (TQ) for 3 weeks. Values are average of five mice.</p

Histopathology of the liver tissues.

<p>(A) Control where BALB/c mice were not injected with WEHI-3 cells. This section shows normal hepatocytes (white arrow) and central veins (green arrow). (B) BALB/c mice injected with WEHI-3 cells and not treated. The leukemia sectioning shows that the hepatocyte cells were mostly destroyed (black arrows), and the Kupffer cells became large (yellow arrows). (C) BALB/c mice injected with WEHI-3 cells and treated with 100 mg/kg thymoquinone. Sectioning showed improvement of hepatic histology over the leukemia, which can be seen similar to the control. (Yellow arrows indicate kuffer cells, white arrows indicate normal hepatocytes).</p