ABSTRACT- Researches on PC-3 human prostate cancer cell lines control are needed in the present decades. The anticancer activity of aqueous extract of Ipomoea Ipomoea sepiaria was investigated by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium (MTT) assay using PC-3 cell line. The present experimentation was showed that aqueous extract of Ipomoea Ipomoea sepiaria, when subjected to different concentrations on PC-3 cells showed IC50 cell inhibition at about 5然 for 48 hours and about 2然 for 72 hours. PTEN interacts with other expression proteins like Jun proto-oncogene, V-akt murine thymoma viral oncogene homolog 1 and 2, Tumor protein p53, Phosphatidylinositol-4,5-bisphosphate 3-kinase, etc. The present experiment shown that the leaf extract may involve in protein suppressor mechanism for PTEN in controlling of prostate cancer.
Key-words- PC-3, Ipomoea Ipomoea sepiaria, Anticancer activity, MTT assay, Prostate cancer

INTRODUCTION
One of the source plants of the classical herb Lakshmana is Ipomoea Ipomoea sepiaria Koenig Ex. Roxb belongs to the family Convolvulaceae. Ipomoea Ipomoea sepiaria (I. Ipomoea sepiaria), a perennial climber is an important ethanomedicinal plant having phyto-constituents like alkaloids, carbohydrates, flavonoids, glycosides, saponin, tannin and phenolic compounds. PC3 (PC-3) is a human prostate cancer cell lines that are highly used in investigating the biochemical changes prostatic cancer cells ^[1-2]. From the last few years, Phytotherapy (herbal therapies) usage for prostate cancer has been increasing dramatically. Several herbs like Chrysanthemum morifolium ^[3], Ganoderma lucidum (a root fungus) ^[4], Glycyrrhiza glabra (Spanish liquorice) ^[5], Scutellaria baicalensis ^[6], Panax pseudoginseng ^[7], Dendranthema morifolium ^[8], Rabdosia rubescens ^[9], and Isatis indigotica ^[10] are tested effective in the treatment of PC-3 cell lines. There were no reports for using Ipomoea Ipomoea sepiaria as a medicinal plant that was used against PC-3 cell line for treating prostatic cancer.
A candidate tumor suppressor gene, PTEN (putative protein tyrosine phosphatase) gene is considered as the responsibility of causing prostate cancer ^[11].
Research about risks and benefits of human prostate cancer screening and treatments are needed in the present decades ^[12]. Protein interaction studies provide better clues in understanding control mechanisms involved in phytotherapy.

MATERIALS AND METHODS
Collection of plant material- Fresh leaves of Ipomoea Ipomoea sepiaria were collected from surrounding areas of Visakhapatnam, India during March 2017 and the work is conducted at Department of Biotechnology, GITAM University, Visakhapatnam, India. The dust particles were removed by washing leaves of Ipomoea Ipomoea sepiaria with double distilled water. The leaves were shade dried and then grounded to powder using mortar and pestle. The obtained powdered samples were then stored in an airtight closed bottle and were used for further experiments.

Preparation of plant extract of Ipomoea Ipomoea sepiaria- About 20gms of the powder of Ipomoea Ipomoea sepiaria plant leaves were taken in 250 ml Erlenmeyer flask. The material was boiled with 100 ml of double distilled water, filtered with Whatman Filter paper No. 1 after cooling and was stored at 4蚓 for further experimentation.

Anticancer Activity of Ipomoea Ipomoea sepiaria against PC-3 cell lines using MTT Assay - To investigated the in vitro inhibitory effects of the aqueous extract from leaves of Ipomoea Ipomoea sepiaria,PC-3 procured from NCCS, Pune, India and sensitivity of PC-3 to Ipomoea Ipomoea sepiaria were determined by the MTT colorimetric assay. About 5000 to 10000 cells approximately in 100 痞 MEM media (MEM199, Sigma, India) per well was seeded in a 96 well plate and incubated at 37蚓, 5% CO₂ for 72 hours. The cells were exposed to leaf extract Ipomoea Ipomoea sepiaria at 6 concentrations 0然, 1然. 2然, 5然, 10然 and 20然. The cells were then treated with, 20痞 of freshly prepared MTT reagent (5mg/ml in PBS) was added and then DMSO (200 痞) was added to each well to dissolve the formazan crystals. The absorbance (OD) of the culture plate was read at a wavelength of 492 nm on an ELISA reader, AnthosBiochrom 2020 ELISA Reader. The percentage of residual cell viability were determined based on the absorbance (OD) obtained from ELISA Reader.

String- String database (https://string-db.org/) is applied for the investigation of protein interaction (PTEN) in human.

RESULTS AND DISCUSSION
Anticancer activity of Ipomoa Ipomoea sepiaria against PC-3 cell line MTT assay - The anticancer activity of aqueous extract was investigated by MTT assay. The present experimentation showed that aqueous extraction of Ipomoea Ipomoea sepiaria when subjected to different concentrations on PC-3 cells were showed IC50 cell inhibition of at about 5然 for 48 hours and about 2然 for 72 hours (Table 1, Fig 1).

Fig. 1: Cultures of PC-3 cells for 48hours Ipomoea Ipomoea sepiaria extract treatment

Table 1: Evaluation cytotoxic property of the extract on PC-3 cells by MTT assay

Time	Concentration	Raw data A1	Raw data A2	Raw data A3	Average	SD	% Cell survival
48h	Control	1	1	1.07809	1.03	0.05	100
	1然	0.9	1	1	0.97	0.06	89.5
	2然	0.6	0.6	0.7	0.63	0.06	58.6
	5然	0.59698	0.6005	0.65	0.62	0.03	57
	10然	0.5	0.4	0.5	0.47	0.06	43.2
	20然	0.2	0.4	0.36625	0.32	0.11	29.8
72h	Control	1	0.9486	1.00259	0.98	0.03	100
	1然	0.8	0.9	0.8	0.83	0.06	85.0
	2然	0.6	0.5	0.50026	0.53	0.06	54.4
	5然	0.5	0.4	0.4	0.43	0.06	44.2
	10然	0.3	0.4	0.2	0.30	0.10	30.6
	20然	0.2	0.2	0.3	0.23	0.06	23.8

Control = mononuclear cells isolated from buffy coats and PC-3 cells


Fig. 2: String database for protein interaction studies for PTEN

Fig. 2 was shown that PTEN interacts with other expression proteins like Jun proto-oncogene, V-akt murine thymoma viral oncogene homolog 1 and 2, Tumor protein p53, Phosphatidylinositol-4, 5-bisphosphate 3-kinase etc. The leaf extract may show protein suppressor mechanism for PTEN in controlling of prostate cancer.
Prostate cancer is a leading cause of cancer death, in humans that was related to the metastatic disease caused due to mutations and different gene expressions ^[13]. Inactivation of PTEN/MMAC1 can cure human prostate cancer through loss of expression mechanism ^[14].
PC3 (PC-3) are prostate cancer cell lines that are used extensively in prostate cancer research ^[15]. Many successful drugs from natural products are acting as an important source for the isolation and activities as anti-cancer lead molecules ^[16]. The molecules may lead to the control and cure of diseases involved in ageing diseases.
Uncontrolled growth due to external factors and internal factors within the biological systems that finally results in death of cells or stopping functionality of components within systems is termed as cancer. Plants have many phytoconstituents like alkaloids, flavonoids, coumarins, polyphenols etc., possesses good antitumor properties ^[17-18].
In the present decades, in silico docking approaches are providing exploring information through the studies of physicochemical characteristics like angiogenesis, diabetes, growth and repair of cancerous cells. Alkaloids from biological sources have good inhibitory effect on cancer cell proliferation ^[19-20].
MTT assay is based on the enzymatic reduction of the tetrazolium salt MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazoliumbromide] for the quantitation measurements of growth modulating effects on the cultured prostate cancer cell lines. The MTT test provides easy and high degree of accuracy measures, hence the test is suitable for large scale purpose of chemosensitivity testing ^[21].

Table 2: Previous studies conducted by MTT assay from Medicinal Plants

Medicinal Plant	Cell line	Reference
Solanum nigrum	HeLa and Vero cell	line Patel et al. [22]
Garcinia mangostana	SKBR3 human breast adenocarcinoma cell line	Moongkarndi et al. [23]
W. somnifera	NCI-H460 (Lung), HCT-116 (Colon), SF-268 (Central Nervous System) and MCF-7 (Breast) human tumor cell lines	Jayaprakasam et al. [24]
Crocus sativus L	HepG2 and HeLa cell lines	Tavakkol-Afshari et al. [25]
Phaleria macrocarpa (Scheff.) Boerl	esophageal cancer cells (TE-2)	Faried et al. [26]
Piper longum	Hep-2	Jacob et al. [27]

Table 2 was shown the usage of medicinal plants acting as anti-cancer agents hat were analyzed using MTT assay. The experimentation of Ipomoea Ipomoea sepiaria against MCF-7 cell lines were previously experimented by Sudhakar and Kaladhar, 2017 [28]. Experimentation on anticancer activity of aqueous leaf extract from Ipomoea Ipomoea sepiaria by MTT assay using PC-3 cell line has been not reported till date.

CONCLUSIONS
The present study has shown that an aqueous extract of Ipomoea Ipomoea sepiaria had considerable anti-cancer activity against prostate cancer cell lines. In silico protein interaction studies may proposed the activation of phyto-compounds from Ipomoea Ipomoea sepiaria may control PTEN molecule that was involved in prostate cancer. These results have shown us a path to conduct in vivo experiments to evaluate the extract of Indian Ipomoea Ipomoea sepiaria.

ACKNOWLEDGMENT
The authors would like to thank JNTUH, GITAM University and Bilaspur University for providing necessary facilities, financial support and technical assistance in bringing out the research work.

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How to cite this article: Meesala S, Rentala S, Kaladhar DSVGK: Anti-cancer Activity of Leaf Extract Preparation from Ipomoea Ipomoea sepiaria against PC-3 Cell Line. Int. J. Life. Sci. Scienti. Res., 2017; 3(5): 1295-1299. DOI:10.21276/ijlssr.2017.3.5.5 Source of Financial Support:Nil, Conflict of interest: Nil