Visnagin inhibits cervical cancer cells proliferation through the induction of apoptosis and modulation of PI3K/AKT/mTOR and MAPK signaling pathway

2.1 Culturing of HELA and Vero cell lines

HeLa (Human cervical cancer cell line) and Vero (Animal kidney cell line) were procured from ATCC, Manassas, USA. Both the HeLa and Vero cells were cultured in EMEM with 10% FBS in 5% CO₂ humidified atmosphere at 37 °C. Cells were replaced with new culture medium for every 48 h and the cells were subcultured upon attaining 80% confluence using 0.25% trypsin-EDTA solution.

2.2 Ferric reducing antioxidant potential assay

FRAP assay was performed according to the protocol of Benzie & Strain, 1996 to quantify the antioxidant potency of visnagin. FRAP reagent was freshly prepared with 300 mM acetate buffer, pH 3.6, 10 mM TPTZ in 40 mM hydrochloric acid and 20 mM ferric chloride in the ratio 10:1:1 respectively. 10 µl of different concentrations of visnagin samples ranging from 1 µM − 100 µM were mixed with 360 µl of FRAP reagent and incubated at 37 °C for 10 min. After incubation the absorbance of the samples were measured at 593 nm using spectrophotometer. Distilled water is used as blank sample and the standard curve is prepared using various concentrations of Trolox ranging from 0.2 to 0.8. The experiments were performed in triplicates. The FRAP value can be calculated as $$\text{FRAP value}(\mu \text{mol TE}/g\text{DW)}=c\times V\times t/m$$

where c – Concentration of Trolox in µmol/ml, V – sample volume in ml, t - dilution factor, and m – dry weight of sample in g.

2.3 DPPH assay

Free radical scavenging property of visnagin was assessed with DPPH radicals using the method of Miliauskas et al. (2004). Visnagin stock solution was prepared with methanol.

Fresh DPPH was prepared by dissolving 7.89 mg of DPPH with 100 ml of 99.5% methanol solution. Before utilizing for the experiment the DPPH solution was kept in dark for 2 h. To a test tube 1000 µl of DPPH solution and 800 µl of Tris-HCl buffer (pH 7.4) were added. To this mixture 200 µl of different concentrations of visnagin ranging from 1 µM − 100 µM was added and kept at 37 °C for 30 min. The OD values of the mixture were measured at 515 nm. Methanol was used as blank. Inhibition ratio percentage was calculated using formula $$\%\text{of inhibition ratio}=\left(\text{A}1-\text{A}2\right)\text{x}100/\text{A}1,$$

A1 - absorbance of blank, A2 - absorbance of test samples.

IC50 was calculated by using interpolation method by selecting two points enclosing 50% inhibition ratio and a regression line of Y = AX + B was drawn. The sample concentration was calculated by substituting 50 in Y value of the above regression equation

Trolox equivalent antioxidant capacity of the samples were calculated using the formula $$\text{TEAC}=\text{IC}50\text{of Trolox}\left(\mu \text{g}/\mathrm{l}\right)/\text{IC}50\text{of test sample}\left(\mu \mathrm{g}/\mathrm{l}\right)$$

2.4 Chemiluminescence assay

Different concentrations of visnagin solution were prepared with 0.1 M phosphate buffer, pH7.4 for the chemiluminescence assay. 0.1 M phosphate buffer was used as blank solution. To10µl test sample or blank, luminol solution was added (1.13x10^-4 M concentration) and then hydrogen peroxide solution was added to make up the final concentration to 5x10^-5 M. 0.2 IU/ml of HRP reagent was added and the chemiluminescence was measured for 10 min at 25 °C. The TEAC of the sample was calculated same as done for the DPPH assay (Krol et al., 1994).

3.1 Cell viability assay

6x10⁴ cells/well of HeLa and Vero cell lines were seeded on to 96 well cell culture plate with DMEM culture medium and grown in 5% CO₂ for overnight. Then cells were treated with visnagin (1 µM-100 µM concentration) and subjected to 24 h incubation. After incubation period 50 µl of MTT solution was added to the wells and incubated for 3 h in 5% CO₂ incubator. The formazan crystals formed due to MTT reaction was dissolved with 100 µl of DMSO and absorbance of solution was measured at 540 nm. $$\text{Percentage of viable cells}=\text{OD of test}/\text{OD of control}\times 100\%$$

3.2 TBARS & antioxidant assay

The TBARS, SOD and GSH assay were done using the commercially available colorimetric kit procured from Abcam, USA. HeLa cells were treated with 10 µM and 25 µM, incubated at 37 °C in 5% CO₂ for 24 h. After incubation the cells were collected, sonicated and utilized for the current experiment. The experiments were performed according to the standard manufacturer’s protocol provided in the kit. The absorbance of the samples was measured using ELISA spectrophotometer. The standard graph was drawn using the absorbance of known concentration. The experiments were performed in triplicates.

3.3 ROS staining

The levels of ROS generated by visnagin against HeLa carcinoma cells were detected using 2′,7′-dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining. After 24 h incubation fo HeLa cells with 10 µM, 25 µM the cells were subjected to DCFH-DA staining. The cells were stained with 10 µM DCFH-DA stain and incubated in dark for 30 min. After incubation the cells were washed with PBS twice and viewed under fluorescent microscope (Olympus).

3.4 AO/ETBr staining

The induction of apoptosis by visnagin on HeLA cells were examined with AO/EtBr staining technique. Vianagin treated cells were stained with mixture of acridine orange and ethidium bromide which was prepared in equal ratio and kept in dark for a period. The stained cells were rinsed with PBS and then viewed under fluorescent microscope.

3.5 PI staining

HeLa cells were treated with 10 µM and 25 µM, incubated at 37 °C in 5% CO₂ for 24 h. The cells were then stained with Propidium Iodide stain for 20 min incubated in dark. The stained cells were rinsed with PBS and then viewed under fluorescent microscope.

3.6 Scratch wound assay

HeLa cell were seeded on to 6 well culture plates and incubated for 24 h, upon attaining 90% confluency the wells were scratched with a sterile 1 ml micropipette tip. The uniformity of scratch was maintained among all the wells. The cells were rinsed with DMEM medium to remove the scratched cells and treated with 10 µM and 25 µM, incubated at 37 °C in 5% CO₂ for 24 h. After incubation the wound closure was viewed microscopically and photographed for further analysis (Liang et al., 2007).

3.7 qPCR analysis

Visnagin treated HeLa cell lines were subjected to qPCR analysis. Total RNA was isolated with TRI reagent procured from Sigma Aldrich, USA. 1 ml of TRI reagent was added to the cells and the cells were scrapped gently with the pipette to obtain a homogenous cell lysate. The cells lysate was sonicated and then 0.2 ml of chloroform was added. The mixture was subjected to 12000rmp for 10 min at 4 °C for phase separation. Aqueous phase was then collected and to that equal volume of ice cold propanol was added, centrifuged at 12000rmp for 10 min at 4 °C for precipitation of RNA. RNA pellet was collected and washed twice with 70% ethanol. RNA pellet was air dried and dissolved in 20 µl milliQ water. RNA purity was examined with NanoDrop spectrophotometer and subjected to complementary DNA synthesis with HiScript II Q RT SuperMix kit.

PI3K, Akt, mTOR, ERK1/2, p38, JNK1/2 gene expression were quantified by amplifying the above genes using SYBR green master mix. The results were analyzed using comparative CT method (2−ΔΔCt) and the fold change of samples was calculated using CFX Manager Version 2.1 (Bio Rad, USA).

3.8 Statistical analyses

All the experiments were performed in triplicates and the data were analyzed statistically using statistics software GraphPad Prism. The data were analyzed with One Way ANOVA followed by the Student Newman Keuls test. The results were expressed as mean ± SEM, and p < 0.05 was considered to statistically significant.

4.1 Antioxidant potency of furanochromone visnagin

In present study the antioxidant potency of furanochrome visnagin was assessed with four different assays and trolox equivalent (TE) values were shown. Fig. 1A depicts the results of Ferric reducing antioxidant potency of visnagin. The TE value of visnagin was significantly increased in a dose dependant manner, maximum of 75 mM TE value was obtained with 100 µM visnagin concentration. DPPH assay results of visnagin were illustrated in the Fig. 1B. The maximum of 7.2 ± 0.8 mM TE value was obtained at 100 µM visnagin concentration. Fig. 1C represents the results of chemiluminescence assay of visnagin. Control shown 1.8 mM TE value during chemiluminescence whereas it significantly increased in different concentrations of visnagin and 100 µM visnagin shown 7.8 mM TE value. Oxygen Radical Absorbance Capacity of visnagin was analyzed with ORAC assay and the results were depicted in the Fig. 1D. Control exhibited 2 mM TE value and the highest concentration 100 µM shown TE value of 17 mM.

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Fig. 1

Antioxidant potency of furanochromone Visnagin. A- Ferric reducing antioxidant potency of visnagin. B - DPPH assay results of visnagin. C- Chemiluminescence assay results of visnagin. E - Oxygen Radical Absorbance Capacity of visnagin. The data were statistically analyzed with One Way ANOVA followed by Student’s Newman Keuls test. The results were expressed as mean ± SEM. p < 0.05, statistically significant.

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4.2 Cytotoxic potency of furanochrome visnagin

Fig. 2A illustrates the MTT results of visnagin treated HeLa cells. The cytotoxic potency visnagin increased in dose dependant manner and about 80% of dead cells were viewed in 100 µM visnagin treated cells. 50% of cell death was observed with 10 µM visnagin treatment. To compare the cytotoxic effect of visnagin in normal epithelial cells, visnagin was treated Vero kidney epithelial cells (Fig. 2B). Even the highest concentration 100 µM visnagin treatment shown only 10% of cell death and only 6–8% of cell death was observed in 10 µM & 25 µM visnagin treatment which shown 50% cell death in HeLa carcinoma cells.

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Fig. 2

Cytotoxic potency of furanochrome visnagin. The cytotoxic potency of visnagin on carcinoma cells and normal epithelial cells were assessed with MTT assay. A – Human cervical carcinoma HeLa cells. B – Animal normal epithelial cells Vero cell line. Results were subjected to statistical analysis with One Way ANOVA followed by Student’s Newman Keuls test. The values does not share a common superscript and significantly differ at p < 0.05.

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4.3 Effect of furanochrome visnagin on antioxidant system in HeLa cells

Visnagin significantly increased the levels of TBARS in HeLa which is the indicator lipid peroxidation in cells. Compared to 10 µM visnagin, the 25 µM visnagin treatment shown drastic increase in TBARS level (Fig. 3A). Increased in antioxidant levels is also key factor for induction of cancer cell induction. Visnagin treatment decreased enzymatic antioxidant superoxide dismutase (Fig. 3B) and non enzymatic antioxidant glutathione (Fig. 3C) levels. The reduction in antioxidant levels was significant and it was in dose dependent manner.

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Fig 3

Effect of furanochrome visnagin on antioxidant system in HeLa cells. HeLa cells were treated with 15 µM and 25 µM visnagin for 24 h, assessed for A- Thiobarbituric acid reactive substances (TBARS), B- Enzymatic activity of superoxide dismutase (SOD), C- Level of non-enzymatic antioxidant glutathione (GSH). The assays were performed with commercially available colorimetric kits as per the manufacturer’s protocol. The results demonstrated that visnagin treatment effectively improved the TBARS content, decreased the SOD activity and GSH level in the HeLa cells. Results were subjected to statistical analysis with One Way ANOVA followed by Student’s Newman Keuls test. p < 0.05, statistically significant, respectively.

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4.4 Free radical generation of furanochrome visnagin on HeLa cells

Fig. 4 illustrates the results of DCFH-DA stained HeLa cells which shows the ROS generation induced by visnagin. Increased green fluorescence emission was observed in visnagin treated HeLa cells compared to the control cells which indicate the generation of free radicals by visnagin treatment. Compared to 10 µM visnagin treatment, the higher dose 25 µM visnagin treatment shown increased ROS generation.

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Fig 4

Free radical generation of furanochrome visnagin on HeLa cells. HeLa cells were treated with 15 µM and 25 µM visnagin for 24 h and assessed for ROS generation by DCFH-DA staining technique. The emission of green fluorescence were viewed and photographed under microscope. The visnagin treated HeLa cells showed a increased green fluorescence, which indicates the substantial increment in the ROS production. Representative images were depicted as Control, 15 µM visnagin treated HeLa cells, and 25 µM visnagin treated HeLa cells, respectively.

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4.5 Apoptotic induction of furanochrome visnagin on HeLa cells

The induction of apoptosis by visnagin on cancer cell line HeLa cells were assessed with AO/EtBr stain and PI staining. AO/EtBr dual staining results were shown in the Fig. 5A. AO/EtBr stain was performed to differentiate live and dead cells in a culture. Acridine orange stain penetrate both the live and dead cells, stains the live cells in green whereas ethidium bromide can only penetrate into the dead cells and stains red. Necrotic cells with normal nuclear morphology will be stained with orange. Our AO/EtBr staining results of visnagin treated HeLa cells shown increased red fluorescence indicating the presence of increased number of dead cells. The red fluorescence increases in dose dependant manner.

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Fig. 5

Apoptotic induction of furanochrome visnagin on HeLa cells. HeLa cells were treated with 15 µM and 25 µM visnagin for 24 h, assessed for apoptotic induction with A- AO/EtBr dual staining technique, B- Propidium iodide nuclear staining technique. The dual staining revealed the increased yellow and orange fluorescence in the visnagin treated HeLa cells. The PI staining images revealed the increased red fluorescence in the visnagin treated HeLa cells. These findings indicates the increased apoptotic cell death. The emission of green and red fluorescence were viewed and photographed under microscope. Representative images were depicted as Control, 15 µM visnagin treated HeLa cells, 25 µM visnagin treated HeLa cells, respectively.

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Fig. 5B depicts the representative propidium iodide stained images of control and visnagin treated HeLa cells. Propidium iodide is a nuclear stain which is permeable only in dead cells and emits red fluorescence. Visnagin treatment increased the cell death in dose dependant manner which is depicted by the increased red fluorescence emission in both 10 µM visnagin and 25 µM visnagin treated HeLa compared to the control untreated HeLa cells.

4.6 Effect of furanochrome visnagin on cancer cell migration

Migration is a key event of cancer cell progress, hence targeting the cancer cell migration with a drug can effectively inhibit the progression of cancer. Fig. 6 represents the inhibitory potency of visnagin on HeLa cells migration assessed with scratch wound assay. Compared to control cells the visnagin treated HeLa cells decreased cell migration. The number of migrated cells in the scratched area is less in visnagin treated group than the control untreated cells.

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Fig 6

Effect of furanochrome visnagin on cancer cell migration. HeLa cells were treated with 15 µM and 25 µM visnagin for 24 h, analyzed for cell migration using scratch wound assay. The migration of cells in scratched area were viewed and photographed under microscope. The findings revealed that the visnagin treatment effectively decreased the migration of HeLa cells. Representative images were depicted as Control, 15 µM visnagin treated HeLa cells, and 25 µM visnagin treated HeLa cells, respectively.

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4.7 Anticarcinogenic potency of furanochrome visnagin on HeLa cells

PI3K/AKT/mTOR pathway is most important intracellular signaling pathway which regulates numerous functions of cells such cell proliferation, cell quiescence and cancer induction in cells. Fig. 7A depicts the results of gene expression of PI3K/AKT/mTOR pathway signaling proteins which were estimated with real time PCR analysis. Visnagin treatment significantly decreased the gene expression of PI3K, AKT, mTOR proteins compared to the untreated control cells. Visnagin treatment also decreased the extracellular cell proliferation signaling pathway MAPK pathway. Visnagin treated decreased ERK1/2, p38 and JNK1/2 protein compared to the control HeLa cells (Fig. 7B).

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Fig 7

Anticarcinogenic potency of furanochrome visnagin on HeLa cells. HeLa cells were treated with 15 µM and 25 µM visnagin for 24 h, assessed for gene expression of cell cycle signaling protein using real time PCR technique. The visnagin treated HeLa cells demonstrated the decreased expression of PI3K, AKT, and mTOR proteins. The visnagin treatment also decreased the ERK1/2, P38, and JNK1/2 expressions in the HeLa cells. A- PI3K/AKT/mTOR pathway signaling proteins, B- MAPK pathway signaling proteins. Results were subjected to statistical analysis with One Way ANOVA followed by Student’s Newman Keuls test. p < 0.05, statistically significant.

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