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In vitro screening for cytotoxic activity of the Blood Vine plant against selected cancer cell lines.

ABSTRACT

Natural products have historically sewed as a source for cancer chemotherapy agents. The ethno- medicinal value of plants provides evidence of their biological activity that can be further utilized for the drug discovery process. This project investigates the cytotoxic properties of the Blood Vine plant against selected cancer cell lines. The ethnomedicinal use of Blood Vine in cancer treatment by Mayan people is the basis for this study. Three different parts (outer, middle and inner) of the Blood Vine stem bark were extracted with ethanol/ water. The extracts were screened for cytotoxic activity against five mammalian cancer cell lines using the WST-1 cell proliferation assay. Ethanolic extracts of the Blood Vine bark have shown potent cytotoxicity against a mouse leukemia cancer cell line (L1210), a mouse hybridoma cell line (7TD1), a pancreatic cancer cell line (PANC1), a glioblastoma cell line (U87) and a breast cancer cell line (SK-BR-3). Results demonstrate that this Blood Vine may be an important source for novel anticancer agents.

Key words: Cytotoxicity, cell proliferation assay, natural products

INTRODUCTION

In 1960, the National Cancer Institute and the United States Department of Agriculture began a screening program for novel antitumor agents from natural products (NP) resulting in the eventual incorporation of various NP-derived drugs into the field of cancer chemotherapy (3), Prior to the 1960's, intensive research in the field of cancer chemotherapy embraced the concept of screening natural products because of the broad structural diversity of NP's. Since then, synthetic combinatorial chemistry has dominated the interest of most major pharmaceutical companies. In recent years, however, NP's have enjoyed a resurgence of interest as a source of anti-cancer agents. This has been in no small part due to greater structural diversity of NP's vis-a-vis purely synthetic drugs. This advantage also translates into greater demand for NP's as scaffolds in the synthesis of novel compounds. NP's derived from microorganisms, plants, animals and microorganisms have delivered many drugs in the field of cancer research since 1960 (6).

Drug discovery from plants is a multi-disciplinary approach which combines various botanical, ethno-botanical, phytochemical and biological and chemical separation techniques (5). Lead identification, lead optimization, and lead development are three main steps of the drug discovery process. Various approaches have been proposed to identify lead compounds from NPs. A very common approach is the consideration of the ethnomedicinal value of NP's. Ethnomedicines are NP's used by indigenous peoples to treat various diseases. The value of ethnomedicine as a source for drug discovery leads lies in the fact that they have been prescreened for bioactivity by the indigenous users. In contrast, only 1-3% of a random collection of NP's are typically bioactive (1).

A well accepted approach in the drug discovery process to identify anti-cancer activities of novel NP's is to measure cell cytotoxicity. A typical way to evaluate cytotoxicity is to use cell-based bioassays with the NP extracts. We have used the WST-1 cell proliferation assay because it is among the most reliable and easiest to perform. This in vitro proliferation assay monitors the conversion of WST-1 dye into formazan in the mitochondria of viable cells.

This research work investigates the cytotoxic activity of an ethnomedicinal plant locally known as Blood Vine obtained from the Guatemalan rainforest. Blood Vine is native to Guatemala and is known by the indigenous peoples to have medicinal properties ranging from treatment of colds to cancer treatment. Currently, the Blood Vine plant is used by Guatemalan people to treat various types of cancer and hence, we hypothesized that the Blood Vine plant inhibits cancer cell growth in vitro. The ethanolic extracts of Blood Vine plant were tested for cytotoxicity against five types of different cancer cell lines.

MATERIALS AND METHODS

Plant Material

The plant samples of Blood Vine were collected by Dr. Richard Hansen from the rainforest of the northern department of Peten, Guatemala. The dried samples have been deposited at Idaho State University. The dried trunk and stem sections of the Blood Vine plant were chosen for examination based on reported ethnomedicinal use. The dried stem of the plant was divided into three parts; outer (OB), middle (MB), and inner bark (IB). The separated sections of the bark were ground into fine powders with an electric grinder.

Preparation of extracts

Powdered samples were extracted into 95% ethanol using a soxhlet extractor. The air-dried powder is placed inside an extraction thimble which is loaded into the extractor. The extractor is mounted inside the flask containing the solvent which is heated to reflux. The whole system is equipped with a condenser. The refluxing solvent repeatedly washes the solid, extracting the desired compound into a flask. Ethanolic extracts were prepared by extracting 1 gram of each type of bark at 78.4[degrees]C for 12 hours. The extracts were evaporated by rotary evaporation and the thin film was dissolved in ethanol:water (4:1). The extracts were named according to the type of bark and name of the solvent (Ex. for ethanolic extracts of inner bark IBET is used). All the extracts were filtered; then centrifuged and resulting supernatant solutions were used to conduct experiments. All the extracts were stored at -20[degrees]C until use. The powdered Blood Vine was stored at room temperature until needed.

The cancer cell lines

Five cell lines were purchased from the American Type Culture Collection (USA): a mouse derived lymphocytic leukemia cell line (L1210), human adenocarcinoma cell line (SK-BR-3), human epithelioid carcinoma (PANC-1), human glioblastoma cell line (U87), and a murine hybridoma cell line (7TD1). L1210 and U87 were maintained in Dulbecco's Modified Eagle's Medium (American Type Culture Collection, USA) supplemented with 10% v/v fetal bovine serum (Atlanta Biologicals, USA). SK-BR-3, PANC-1 and 7TD1 cell lines were maintained in RPMI- media (American Type Culture Collection, USA) supplemented with 10% v/v fetal bovine serum (Atlanta Biologicals, USA). All the cell cultures were incubated at 37[degrees]C in a tissue culture incubator (Thermo Corporation) with a 5% v/ C[O.sub.2] and 95% relative humidity.

In vitro determination of anticancer activity

Blood Vine extracts were diluted and were labeled according to dilution strength (Example: ethanolic extracts of inner bark with 1,5 and 50 times dilution were labeled as IBET1X, IBET5X and IBET50X respectively). The cytotoxicity of extracts was tested using the WST-1(Clonetech, CA, USA) cell proliferation assay. L1210 and 7TD1 were seeded at 5x[10.sup.4] cells/mL into 96-well flat bottom microplates (Costar, USA). SK-BR-3, PANC-1 and U87 were seeded at 10x[10.sup.3] cells/mL. All the cells were incubated as mentioned above. After 24 hrs, 5 [micro]L of treatment solution was added into each of the 4 wells per treatment. The treatments were vehicle control, positive control and ethanolic extracts of Blood Vine to each of 4 wells. The cytotoxic activity of ethanolic and water extracts were compared against positive control and vehicle control. 10 [micro]M solutions of adriamycin, etoposide, cisplatin and rapamycin were used as a positive controls. Ethanol:water (4:1) was used as a vehicle control. The treated cultures were incubated for 72 hrs and 10 [micro]L of WST-1 dye was then added to each well and the plate vigorously agitated for 60 s to ensure mixing of the dye with the cell culture. Absorbance measurements at 440 nm were made immediately after adding the WST-1 dye and following additional 4 hr incubation at 37[degrees]C. The average absorbance from each of the 4 wells of each treatment at each time point was calculated as:

[([Avg abs.sub.t4hr] - [Avg abs.sub.t0]).sub.vehicle] / [([Avg abs.sub.t4hr] - [Avg abs.sub.t0]).sub.treatment]

RESULTS

All parts of the Blood Vine showed potent cytotoxic activity against the selected cancer cell lines. The outer bark of Blood Vine showed the most prominent cytotoxicity against the L1210 cell line (Figure 1). The outer bark, middle bark and inner bark were equally potent against SK-BR-3 cells (Figure 2). In the case of PANC1 cells, the inner bark showed the most potent cytotoxic activity (Figure 3). In the case of U87 cells, the outer bark was the most potent cytotoxic agent (Figure 5). The outer and middle bark showed similar cytotoxicity against 7TD1 cells (Figure 4). The outer bark ethanolic extracts of Blood Vine inhibited the growth of almost all cancer cell lines mentioned above.

Every type of cancer is treated by different anticancer agents specific to type of cancer hence, various positive controls (anticancer agents) are used for different type of cancer cells while studying the bioactive activity.

DISCUSSION

Although cancer mortality rate is declining, cancer remains a challenging field for researchers. While various conventionally derived synthetic drugs and drugs derived by combinatorial chemistry are now available in the market (4), the development of new anticancer agents with novel chemical structures continues to be a difficult endeavor. The structural diversity of NP's will continue to offer new and original options to drug development (2).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

The current research work follows the typical process of drug discovery from natural products, i.e. identification of bioactivity, isolation, purification and structural elucidation of active compounds. This project investigated an ethnomedicinal plant obtained from the Guatemala rainforest which has shown potent cytotoxicity against five types of cancer cell lines. However, the activity has not been confirmed in vivo. The outer bark of the Blood Vine has shown consistent cytotoxic activity against all the selected cancer cell lines. Consequently, refinement of outer bark extract is currently being pursued using High Performance Liquid Chromatography (HPLC) (data not shown) and the structures will be determined with the help of Mass Spectrometry(MS) and Nuclear Magnetic Resonance (NMR). Inner and middle bark extracts will be similarly examined. While a full phytochemical profiling of the extract is outside the scope of this work, a comparison of active compound(s) in each bark section along with relative abundances will be pursued. It is hoped that this work will result in one or more novel lead compounds for anti-cancer drug development.

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

In addition, studying a reason behind a pronounced cytotoxic activity of outer bark will be an interesting part of the project. Investigation of mechanism of action of outer bark, middle bark and inner bark will be one of the striking features of this project.

The ultimate aim of the project is to demonstrate the value of Blood Vine and similar ethnomedicinal plants from the Guatemalan rainforests, which are currently threatened by expanding human development.

ACKNOWLEDGEMENTS

Dr. Richard Hansen for the Blood Vine samples.

Dr. Dana Diedrich for his expert advice.

College of Pharmacy, the ISU Office of Research for financial support.

INBRE for financial support

REFERENCES

(1.) Christian Bailly, 2009. Ready for a comeback of natural products in oncology, BIOCHEMICAL PHARMACOLOGY, VOL 77, 1447-1457.

(2.) Daniel S. Fabricant and Norman R. Farnsworth,2001. The Value of Plants Used in Traditional Medicine for Drug Discovery. Environmental Health Perspectives, Vol 109,supplement 1

(3.) Douglas Kinghorn et.al, 2003. Novel Strategies for the Discovery of Plant-Derived Anticancer Agents. Pharmaceutical Biology, Volume 41, Supplement, pp.53-67

(4.) Kuo-Hsiung Lee, 2004. Current Developments in the Discovery and Design of New Drug Candidates from Plant Natural Product Leads. Journal of Natural Products, vol 67, 273-283.

(5.) Sanjay M. Jachak and Arvind Saklani 2007. Challenges and opportunities in drug discovery from plants. CURRENT SCIENCE, vol. 92, No 9

(6.) Young-Won Chin et al, 2006. Drug Discovery From Natural Sources. The AAPS Journal; 8(2)
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Title Annotation:PROCEEDINGS PAPERS FROM THE MEETING
Publication:Journal of the Idaho Academy of Science
Article Type:Report
Geographic Code:1USA
Date:Jun 1, 2010
Words:1915
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