 |
| About Bioline | All Journals | Testimonials | Membership | News |
|
||||||
|
||||||
Indian Journal of Pharmacology, Vol. 37, No. 4, July-August, 2005, pp. 232-237 Research Paper Induction of cell-specific apoptosis and protection from Dalton's lymphoma challenge in mice by an active fraction from Emilia sonchifolia Shylesh B.S., Nair S. Ajikumaran, Subramoniam A. Tropical Botanic Garden and Research Institute, Palode, Thiruvanathpuram Date of Submission: 21-Feb-2005 Code Number: ph05059 ABSTRACT Objective: To isolate an active anticancer fraction from Emilia sonchifolia and to determine the mechanism of its anticancer activity.Materials and methods: The anticancer principle was separated using thin layer chromatography (TLC) from the most active n-hexane extract and chemically analysed. The anticancer efficacy of n-hexane extract was determined in mice using Dalton's lymphoma ascitic (DLA) cells. Cytotoxicity of the extracts and isolates to macrophages, thymocytes and DLA cells was measured using Trypan blue exclusion method, MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay, DNA ladder assay and DNA synthesis in culture. Short-term toxicity evaluation of the active fraction was also carried out in mice. Results: The hexane extract was found to be most active and it showed in vitro cytotoxicity to DLA and thymocytes, but not to macrophages. In a concentration and time-dependent manner, it induced membrane blebbing, nuclear condensation, DNA ladder formation, and formation of apoptotic bodies which are characteristic to apoptotic cell death. The n-hexane fraction protected 50% of mice challenged intraperitoneally with 106 DLA cells. This fraction did not exhibit conspicuous adverse toxic symptoms in mice. An active terpene fraction was separated from the n-hexane extract by TLC. This isolate induced apoptotic cell death in DLA cells at 0.8 µg per mL level. Conclusion: An anticancer terpene fraction was isolated by TLC from Emilia sonchifolia that induced cell-specific apoptosis and appears to be a promising anticancer agent. Keywords: Anticancer herbal extract, terpene, apoptosis, thymocytes, macrophages INTRODUCTION Emilia sonchifolia (L.) DC. ex Wt. (Compositae), is an herbaceous plant found in India and in other Asian countries.[1] The plant is used in folklore medicine for the treatment of tumor, inflammation, cough, rheumatism, cuts, and wounds. In China, the leaves of this plant are used against fever and dysentery.[2] The water extract of this plant showed antimicrobial activity.[3] The aerial part of the plant has been reported to contain alkaloids,[4] flavonoids, and terpenes.[3] Earlier, based on the limited studies, it has been reported that the alcohol extract of this plant (aerial part) has cytotoxicity, as well as anti Ehrlich ascitic carcinoma (EAC) and anti Dalton′s lymphoma ascites (DLA) activities in mice.[5] In the present study, the earlier findings were extended, an active n-hexane fraction (extract) was isolated and the induction of cell-specific apoptosis by the active fraction was discovered. The active principle was separated by TLC and identified as terpene. A preliminary short-term toxicity evaluation of the active fraction was also carried out in mice.MATERIALS AND METHODS Plant materialEmilia sonchifolia, collected locally from Thiruvanantha-puram District, Kerala, was used for the studies. It was identified and compared with the herbarium specimen preserved at the Tropical Botanic Garden and Research Institute (TBGRI) by taxonomists of TBGRI. Preparation of extracts The collected plants were washed thoroughly in tap water, dried in shade, and powdered. The powder was extracted sequentially at room temperature with different solvents: n-hexane, ethyl acetate, ethyl alcohol, butanol and water essentially as described elsewhere.[6] At each step, the extraction was done 3 times with fresh solvent to ensure complete extraction. The extracts were dried in a rotary evaporator at 40 oC except for water extract that was freeze dried. Animals Inbred Swiss albino mice (6-7 week old), reared in the TBGRI animal house, were used. Animals were caged in uniform hygienic conditions and fed with standard pellet diet (Lipton, India Laboratories, Banglore) and water ad libitum. Clearance was obtained from the Institute Animal Ethics Committee to conduct these experiments. Chemicals RPMI-1640 medium, phosphate buffered saline (PBS), streptomycin, and penicillin were purchased from High Media, India Limited. RNA-ase A, proteinase K, 3- [4, 5-dimetylthiazol-2yl]-2, 5-diphenyl tetrazolium bromide (MTT), agarose, ethidium bromide, vincristine, and curcumin were purchased from Sigma Aldrich, St. Louis, USA. Methyl-3H-thymidine was purchased from the Board of Radiation and Isotope Technology (BRIT), Mumbai, India. Cell lines Dalton′s lymphoma ascitic (DLA) cells, originally obtained from Amala Cancer Research Centre, Thrissur, India, were propagated as transplantable tumors in the peritoneal cavity of mice. Thymocyte preparation The thymus glands were removed from the mice carefully and trimmed off from the adjoining lymph nodes. Single-cell suspensions were prepared in cold RPMI -1640 medium and viability assessed by Trypan blue exclusion method.[7] Collection of macrophages Peritoneal exudates cells (PEC) were collected by injecting 5 ml chilled RPMI-1640 medium in peritoneal cavity of mice. The glass adherent cell population (macrophages) was separated by adhering PEC over glass petri-dish at 37 oC for 2 hours in a O2 incubator having 5% O2 in air. The viable cell count was done using Trypan blue in a Neubauer counting chamber. In vitro cytotoxicity assay Short-term cytotoxicity of extracts was assessed by incubating 1 x 106 DLA cells, thymocytes or macrophages in 1 ml PBS containing different concentrations of extract. The cell viability was assessed by the Trypan blue exclusion method.[7] Long-term cytotoxicity of extracts/fractions to DLA cells was determined by seeding 1 x 106 cells in 24-well-plate containing 1 ml RPMI-1640 medium supplemented with 10% FBS (fetal bovine serum), streptomycin (100 µg/ml) and penicillin (100 units/ml). The cells were grown at 37 oC in a humidified atmosphere in 4% O2 and 96% air. The cell viability was determined by the Trypan blue exclusion method.[7] The toxicity was also determined by MTT assay. It was performed essentially as described earlier.[8] Briefly 1 x 10 6 cells in one ml were seeded in 24-well-plate and the cells were exposed to different concentrations of n-hexane extract. After incubation, MTT solution (1.2 mg/ml) was added to each well and cells were incubated for an additional 4 h. The MTT formazan product was dissolved in DMSO and the color was estimated using Elisa plate reader at 570 nm absorbance. 3H-thymidine incorporation assay DLA cells were cultured in RPMI-1640 containing 1 µCi/ml 3H-thymidine. The cells were exposed with different concentrations of n-hexane fraction at 37 oC for 5 h. The radio-activity was measured, as described elsewhere, by liquid scintillation counter.[9] Apoptosis Because membrane blebbing was observed during cytotoxicity assay, a detailed study was carried out to determine whether or not the hexane extract induces apoptotic cell death. Morphological observation and DNA-ladder formation assay were carried out. Morphological evidence was determined by using acridine orange and ethidium bromide (AO/EtBr) staining.[10] Cells (1 x 106) were seeded in 24-well-plate along with various concentrations of n-hexane extract and curcumin (a positive control) at 37oC for 4 hours. The cells were washed with PBS and treated with AO/EtBr (100 µg/ml PBS of each dye). The cells were observed under fluorescent microscopy using a blue filter, and photographed (ASA-400 Kodak); non-viable cells were observed in red-orange color. (The damaged membrane allows EtBr to enter and will appear as red-orange color). DNA ladder assay DLA cells (1 x 106) were seeded in 24-well-plate containing RPMI medium, along with various concentrations of n-hexane fraction and curcumin and incubated for 24 h in 4% O2 and 96% air. After incubation, cells were harvested and washed with PBS and pelleted by centrifugation (300 rpm). To the cell pellet, 100 µL lyses buffer (50 mM Tris -HCl pH 8., 20 mM EDTA, 10 mM NaCl, 1 % SDS) was added for 1 min and centrifuged at 2000 rpm for 5 min. The supernatant was collected and incubated with RNase A (5 µg/ml) for 1 hour followed by digestion of proteins with proteinase K (100 µg/ml) for 5 hours. Aliquots of lysate (20 µL) were loaded to 1.5% agarose prepared in TBE buffer [Tris-Borate 45 mM, 1 mM EDTA, pH 8] containing 3 µg/ml EtBr, and DNA was separated by electrophoresis. The separated DNA fragments in the gel were visualized by the gel-documentation system. Antitumor activity of n-hexane extract in mice Antitumor activity of n-hexane extract was determined, as described previously.[5], [11] Briefly, 48 Swiss albino mice (20-25 g) were challenged with DLA cells (1 x 106 cells, i.p) and divided into 6 groups of 8 each. Group I: Served as control, received 1% gum acacia orally. The groups, II, III and IV received the n-hexane extract/fraction of E. sonchifolia 50, 100, and 250 mg/kg, b.w., respectively; the groups V and VI received 0.5 and 1 mg/kg, b.w., vincristine, respectively, orally for 15 days. (Vincristine was used as a standard anticancer agent for comparison). Chemical analysis of n-hexane extract The n-hexane extract was subjected to chemical analysis to determine the classes of compounds present in it.[12] The extract was tested for the presence of alkaloid (Dragendorff reagent and Mayer′s reagent), coumarin (Borntagrs reaction), flavonoid (Shinoda test), steroid (Liberman Burchard test), steroid, and terpene (vanillin- sulphuric acid reagent). The n-hexane fraction was subjected to silica gel thin layer chromatography (TLC) using n-hexane:chloroform:methanol (5:4.5:0.5) as a solvent system. The chromatograms were sprayed with Libermann-Burchard reagent or vanillin- sulphuric acid reagent and heated at 100 oC and inspected.[12] The chromatogram showed several terpenoid and steroid spots. Each spot in preparative TLC was identified (with the help of a reagent sprayed plate run simultaneously) on the basis of relative mobility and scrapped off and eluted with n-hexane and tested for apoptotic cell death induction in DLA cells. Preliminary toxicity evaluation To determine the preliminary toxicity, 3 groups of animals (n = 6) were administered orally, 0 (control), 250 and 500 mg/kg of the n-hexane extract, respectively, for 30 days. The control group received 1% gum acacia in an identical manner. Body weight, food and water intake, and general behavior were observed. After 30 days the animals were sacrificed and blood was collected; organs (thymus, spleen, liver, and kidney) were removed and weighed. Hematological parameters were determined, as described elsewhere.[13] Serum glutamate pyruvate transaminase (SGPT) and serum glutamate oxalate transaminase (SGOT) were estimated by the method of Reitman and Frankel;[14] serum alkaline phosphatase (AP) was estimated by the Kind and King′s method[15] using commercial kit; urea was estimated colorimetrically using commercial kit (Span diagnostics, India); cholesterol was estimated, as described elsewhere;[16] glucose was estimated enzymatically by GOD/POD method. RESULTS Cytotoxicity DISCUSSION In the present study, it has been shown clearly that the n-hexane fraction from E. sonchifolia, as well as the terpene isolated from the extract, induced cell-specific apoptosis. Although there are a lot of herbal extracts or isolated compounds with varying levels of anticancer activity, they are rarely cell specific.[17] Similarly curcumin, a common antioxidant and antiinflammatory agent, also showed comparable cytotoxicity and it was not cell specific. The cytotoxicity of n-hexane extract and terpene isolate from E. sonchifolia were found to be cell specific. The extract was not toxic to macrophages, whereas it was toxic to thymocytes and cancer cells tested in the present study. Earlier preliminary studies have shown that the extract of E. sonchifolia was toxic to Ehrlich ascitic carcinoma (EAC) cells and transformed mouse lung fibroblasts, and not toxic to normal human peripheral lymphocytes.[5] Therefore, it is likely that it could be active against specific types of cancer cells sparing almost all normal cells except sensitive cells like thymocytes. Further, it is of interest to note that a dose much higher than the therapeutic dose did not show severe adverse toxic manifestations in mice. However, the significant reduction in total WBC needs further evaluation. The sensitivity of thymocytes (probably immature T-cells) to the herbal drug may account, at least, partly to this observed reduction in WBC. This may affect the normal function of immune system. The moderate increase in alkaline phosphatase (AP) observed in the toxicity study may suggest minor damage to some cells in one or more organs such as bone and intestine which are rich sources of AP. Because serum transaminase levels were not changed, liver damage and entry of liver AP into the circulation is highly unlikely. Further, normal morphology and weight of the liver were not affected by the treatment.The mild decrease in serum cholesterol level may be considered as a beneficial side effect. The n-hexane fraction and/or the active terpene are attractive materials for drug development. However, a detailed study is required about the sensitivity of different normal cells to the herbal drug under in vivo conditions. It is of interest to note, the in vivo efficacy of the hexane fraction (250 mg/kg) against Dalton′s Lymphoma was equal to vincristine (1 mg/kg). The dose of vincristine used was the maximum-tolerable dose, whereas in the case of the hexane fraction, a higher dose is possible without conspicuous major toxic manifestations. However, there is an urgent need to test the efficacy of this drug in various cancer models. Such studies in this laboratory in various animal cancer models are underway. It is known that antioxidants can inhibit proliferation of at least certain types of cancers.[18], [19] Agents that show antiinflammatory activity by inhibiting cyclo-oxygenase can also retard cell proliferation.[19] Previous studies have shown that the extract of E. sonchifolia has both antiinflammatory and antioxidant properties.[20] It remains to be studied whether or not the antiinflammatory and antioxidant activities are exerted by the apoptosis inducing terpene identified in the present study. It is likely that the antitumour terpene could have more than one molecular target in exhibiting its anticancer action. At any rate, the present study establishes the likely anticancer potential of E. sonchifolia. Studies in progress in this laboratory include structural identification of the terpene active principle, as well as the mechanism of action studies which include the molecular event that triggers the chain of events leading to apoptotic cell death. E. sonchifolia is an annual herb which grows as a weed. This can be easily cultivated, and the plant material can be made available in sufficient quantities as and when needed for drug development. However, the cultivation conditions that yield maximum bio-active material have to be studied. ACKNOWLEDGEMENTS One of the authors, (Dr. B.S. Shylesh) gratefully acknowledges¸ CSIR, Govt. of India, for having provided a Research Associate position to carry out this study. The authors express their sincere thanks to Mr. G. Santhoskumar, Animal House Technician, for providing technical assistance in the execution of animal experiments. Dr. S. Seeni, Head, Dept. of Biotechnology, Mr. P. Padmesh, Scientist, and J.V. Reji, SRF, TBGRI, are acknowledged for their help in the DNA fragmentation analysis. Dr. (Mrs) Prabha Balaraman, Professor, Regional Cancer Research Centre, Trivandrum is gratefully acknowledged for providing photo-micrographic facilities.REFERENCES
Copyright 2005 - Indian Journal of Pharmacology The following images related to this document are available:Photo images[ph05059f1.jpg] [ph05059f3.jpg] [ph05059f4.jpg] [ph05059f2.jpg] [ph05059t2.jpg] [ph05059t4.jpg] [ph05059t1.jpg] [ph05059t3.jpg] |
| |||||||||
