Evaluation of Apoptotic Activity of Ethanolic Extract of Andrographis paniculata Linn in Human Hepatocellular Carcinoma Cell Lines

Introduction: Andrographis paniculata is one of the highly used potential medicinal plants in the world. This plant is traditionally used for the treatment of common cold, diarrhoea, fever due to several infective cause, jaundice, as a health tonic for the liver and cardiovascular health, and as an antioxidant. It is also used to improve sexual dysfunctions and serve as a contraceptive. All parts of this plant are used to extract the active phytochemicals, but the compositions of phytoconstituents widely differ from one part to another and with place, season, and time of harvest. Materials and Methods: All data obtained were analysed by Student t test using MSExcel. The results were computed statistically using one way ANOVA.The reagents used were MTT [3-(4,5dimethythiazol-2-yl) 2,5-diphenyl tetrazolium bromide]: 0.5 mg MTT/ml of serum-free DMEM, Solubilization solution: Dimethyl sulfoxide (DMSO)and Phosphate Buffered Saline (PBS; pH 7.4).The cytotoxic effects of lycopene Ag NP on 3T3 cells. Cells were treated with Lycopene Ag NP(10, 20, 30, 50, 100 and 200 μM) for 24 h, and cell viability was evaluated by MTT assay. Data are shown as means ± SD (n = 3). * compared with the control-blank group, p < 0.001. Original Research Article Kannan et al.; JPRI, 33(64B): 332-341, 2021; Article no.JPRI.77724 333 Results: The results were evaluated using three tests namely, MTT assay, Dual staining (acridine orange/ethidium bromide) and morphological changes. Conclusion: Different stages of apoptosis revealed the importance of A.paniculata in hepatocellular carcinoma cell lines. Andrographolide and analogues have been subjected to extensive chemicobiological investigations for anticancer drug development. Several andrographolide analogues have shown superior anticancer activities in both in vitro and in vivo models. Further clinical and biomedical studies are required to confirm the pharmacological, pharmaceutical and toxicological properties of andrographolide.


INTRODUCTION
Medicinal plant is an integral part of human life to combat the sufferings from the dawn of civilization [1]. It is estimated that more than 80,000 of total plant species have been identified and used as medicinal plants around the world [2]. Liver cancer is that the third leading explanation for cancer death worldwide and regarding half the patients with liver disease need adjuvant medical care when surgical surgical operation [3,4]. Therefore, development of novel agents to eradicate cancer cells might represent a viable approach to treat patients with liver disease [5]. Andrographolide, a diterpenoid lactone isolated from Andrographis paniculata, is understood to possess potent inhibitor, medication, antineoplastic and antiviral properties [6]. During this study, we tend to investigate the cytotoxic impact of andrographolide on human liver disease cells and explore the necrobiosis mechanism [7]. Andrographolide iatrogenic a necrobiosis distinct from cell death in multiple human liver disease cells [8]. The death was characterised by autophagy as proven by the buildup of LC3 II and autophagosomes, and therefore the formation of puncta GFP-LC3 [9-15]. ('Molecular structure and vibrational spectra of 2,6bis(benzylidene)cyclohexanone: A density functional theoretical study ', 2011) [16-28]. This autophagy similarly as toxicity caused by andrographolide [29] may well be effectively prevented by 3-methyladenine (a chemical substance of autophagy) [30,31].
Mechanistic study indicated that andrographolide induced autophagic necrobiosis by disruption of mitochondrial [32] transmembrane potential and elevation of reactive element species [33,34] that were related with mitochondrial porosity transition pore Inhibition of cyclophilin D (a element of MPTP) by cyclosporin A or repeal of its expression by tiny meddlesome ribonucleic acid considerably suppressed the toxicity of andrographolide [35], suggesting that cyclophilin D could play a very important role in mediating andrographolide-induced toxicity [36,37]. Taken along, our findings unveil a unique mechanism of drug action by andrographolide in cancer of the liver cells [38] and counsel that andrographolide could represent a promising novel agent within the treatment of cancer of the liver [39,40]. The aim of this study is to evaluate apoptotic activity of A. paniculata in controlling hepatocellular carcinoma.

DMEM
Commercially available DMEM contains 7.5% sodium bicarbonate solution. To 500ml of DMEM, 5ml of penicillin/streptomycin solution and 0.5ml of amphotericin B solution was added. Then the medium was sterile filtered (0.22μ) inside the hood. The medium was then dispensed into sterile containers and stored at 4°C.

Growth medium (DMEM with 10% FBS)
10ml of FBS was made up to 100ml using sterile DMEM. It was stored in a sterile container in cool and aseptic condition.

Phosphate buffered saline (PBS; pH 7.4)
0.63 g of sodium phosphate monobasic (NaH2PO4), 0.17 g of sodium phosphate dibasic (Na2HPO4) and 4.5 g of sodium chloride (NaCl) were dissolved in 500 ml of double autoclaved milliQ water. The pH was then adjusted to 7.4 using 1 N HCl and 1 N NaOH, sterile filtered (0.22 μ) and then stored in a sterile container.

Trypsin-EDTA
Trypsin was purchased as 1 x with EDTA (0.5% trypsin, 5.3 mM EDTA sodium salt). (Note: Freeze-thaw process does not affect the enzyme activity. Thawing is done at room temperature).

Cell line maintenance
Human breast cancer cell line was procured from the National Centre for Cell Science (NCCS, Pune), India. The cells were grown in T25 culture flasks containing DMEM medium supplemented with 10% FBS. Upon reaching confluence, the cells were detached using Trypsin-EDTA solution.

Passaging of cells
Upon reaching the confluence cell line and were passed as follows.
The medium from the culture flask was aspirated. The flask was rinsed with 2ml of PBS and aspirated again quickly. 1.5-ml of trypsin-EDTA solution was added and incubated at 37ºC for about 2 minutes until cells started detaching. As soon as the cells were detached, transfer the trypsinized medium containing cells using a transfer pipette into a 15 ml falcon tube and it was centrifuged at 1000 rpm for 5 min. The medium was carefully aspirated off and care was taken not to put the pipette tip in the bottom of the tube, where the cells were pelleted. The cells were gently resuspended in the DMEM medium with 10% FBS by pipetting up and down 5-8 times gently. From the cell suspension, a drop was placed on the edge of the coverslip of neubaurer hemocytometer. The drop was led to run under the cover slip by capillary action. Care was taken not to force the liquid and the entry of air bubble was avoided. Then the cells from the E1, E2, E3, E4 and E5 squares were counted under microscope. The cells were then gently resuspended and transferred to sterile culture flasks and the volume of medium was made upto required volume with growth medium per flask.

Principle
The assay is based on the reduction of soluble yellow tetrazolium salt to insoluble purple formazan crystals by metabolically active cells. Only live cells are able to take up the tetrazolium salt .
The enzyme (mitochondrial dehydrogenase) present in the mitochondria of the live cells is able to convert internalized tetrazolium salt to formazan crystals, which are purple in colour. Then the cells are lysed using a 20% SDS solution, which releases the formazan crystal [42]. These crystals are solubilized by DMF present in the solubilizer. The colour developed is then determined in an ELISA reader at 620 nm.

Morphology Study
Based on MTT assay we selected the optimal doses (---) for further studies. Analysis of cell morphology changes by a phase contrast microscope. 3×104 cells were seeded in 6 well plates and treated with Compound Name (concentration for MCF-7 cells) for 24h [45,46]. At the end of the incubation period, the medium was removed and cells were washed once with a phosphate buffer saline (PBS pH 7.4). The plates were observed under a phase contrast microscope.

Ethidium Bromide/Acridine Orange Staining (Ao/Eb Staining Or Dual Staining)
A549/HepG2 cells were plated at a density of 5 x 104 in 6 well plates containing sterile coverslips. They were allowed to grow at 37ºC in a humidified CO2 incubator until they are 70 -80% confluen [43].
Then cells were treated with synthesized (50µM) for 24hrs. The culture medium was aspirated from each well and the cells were gently rinsed twice with PBS at room temperature [43]. Then the coverslips were taken and kept on glass slides and stained with 100 µl of dye mixture

Statistical Analysis
All data obtained were analyzed by Student's-ttest using MS-Excel, represented as mean ± SD for triplicates. The results were computed statistically (SPSS/10 Software Package; SPSS Inc., Chicago, IL, USA) using one-way ANOVA.
The level of statistical significance was set at p<0.05. In study conducted by Wei, we investigated the effects of andrographolide on human liver cancer cells and further examined the cell death mechanism [52]. Our observations demonstrated that andrographolide-induced cytotoxicity was attributed to autophagy but not apoptosis in human liver cancer cells and that this autophagyinducing activity was closely associated with the cyclophilin D-mediated mitochondrial permeability transition pore (MPTP) [53]. The preclinical studies conducted by Karl suggest that andrographolide could be useful for the treatment of liver cancer [54]. Andrographolide suppresses apoptosis of human umbilical vein endothelial cells (HUVECs) induced by growth factor deprivation via the activation of PI3/Akt pathway.

CONCLUSION
The three findings confirm that andrographolide induces apoptosis effectively in HepG2 human hepato cellular carcinoma cells In vitro [54][55][56][57][58][59][60]. Taken together, our findings unveil a novel mechanism of drug action by andrographolide in liver cancer cells and suggest that andrographolide may represent a promising novel agent in the treatment of liver cancer. Further investigations revealed needed at pre-clinical and clinical levels for establishing it as a potential agent for cancer therapy.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.

COMPETING INTERESTS
Authors have declared that no competing interests exist.