INTRODUCTION• Natural compounds are well known as source for drug of several human ailments.• Ex. Vincristine, irinotecan and paclitaxel.• Search for a new anticancer agent is still necessary in order to increase the range available and to find less toxic and more effective drug.• Therefore, this work was design to investigate the cytotoxicity of six natural compounds available in research group, with previously demonstrated pharmacological activities.
• 1. Xanthin V1- roots of Cratoxylum formosum, the leaves of Symphonia globulifera and the seeds of Vismia laurentii• 2. 2 acetylfuro 1,4- napthoquinone- Newbouldia laevis• 3. Physcion- Rheum officinale• 4. Bisvismiaquinone- Vismia genus• 5. Vismiaquinone- Vismia genus• 6. 1,8 dihydroxy-3-generyloxy-6-methylanthraquinone- Vimia laurentii.• In this work these examined the cytotoxicity of 1-6 compounds against MiaPaCa-2, CCRM-CEM, and CEM/ADR-5000 cell lines, and then selected compound 1and 2 to test on a panel of cancer cell lines.
Material and Methods• Cell lines were obtained from different sources.• Cell lines used are- CCRM-CEM (Human T cell Lymphoblast cells) CEM/ADR5000 PF-382 (Leukemia T cells) HL-60 (Premyelotic leukemia) MiaPaCa-2 (Pancreatic adenocarcinoma) Capan-1(Pancreatic adenocarcinoma) MCF-7 (Breast cancer ) SW-680 (Colon Carcinoma) 786-0 (Renal Carcinoma) U87MG (Glioblastoma astrocytoma cells) A-549 (Lung adenocarcinoma) Colo-38 (Skin Myeloma Cells) HeLa (cervical Carcinoma cells) Caski (Cervical Carcinoma Cells) AML 12 (Hepatocytes)
• All culture cells were maintained in a humidified environment at 37 0 C with 5% CO2.• Doxorubicin was used as a positive control.• It is a drug used in wide range of cancer chemotherapy.• Isolation and characterization of six natural compounds used in this experiment has been previously done.
Resazurin Cell Growth Assay• This assay test cellular viability and mitochondrial function.• Adherant cells were grown in Tissue Culture Flask and harvested by treating trypsin and EDTA for 5 min.• Once deteched, Cells were washed, counted and aliquot of 5000 cells was places in each well of 96 well plate in totle vol. of 100 microlitter.• Overnight incubation to allow them to attach.• And then treated with Samples with a range of 20-0.16 microgram/ml.• Incubation for 48 hr.• After 48 hr., 20 microlittre of resazurin was added.• Incubation at 37 degree C for 1-2 hr.• Flourescence was measured on Plate reader using excitation wavelength 544nm and emmission at 590 nm.
• For leukemia cells, aliquot of 50000 cell were seeded in 96 well plate and extract were added immediately.• After 24 hr. plates were treated with resazurin as mentioned before.• Each Assay was done at least 3 times with 2 replicates.• Viability was measured by comparison with untreated cells.
• Sample 1 and 2 as well as doxorubicin were able to reduce he proliferation of 3 cell lines up to 50 % when tested at 20 microgram/ml.• Physcion was found to have no cytotoxicity.• Rest 4, 5, and 6 compounds also bearing physcion moiety, showing the low cytotoxicity.• So the sample 1 and 2 were selected for further effect on diff. cell lines..
• The IC 50 value below 20 microgram/ml were recorded in 12 of the 14 cell lines for sample 1, and 14/14 cell lines for sample 2.• Below or arround 4 mg/ml Sample 1- 9/14 Sample 2- 11/14(IC 50 value= Conc. Of sample required to inhibit 50% of cell proliferation.)
Flow Cytometry for cell cycle analysis and detection of apoptotic cells• CCRM-CEM cells were treated with compound 1 and 2 or DMSO for 24 to 72 hrs.• Then fixed with ethanol 95 %and then washed with cold PBS.• Resuspand in 150 microlitter hypotonic fluorochrome.• Cells were incubated in dark for overnight before FACS.• PI flouroscence was measured of individual nuclei by FACS Caliber.• Data were analyzed by CellQuess Pro V5.2.1.• At least 3 experiments were performed.
Caspase Glo 3/7 Activity• CCRM-CEM leukemia were cultured in RPMI, were seeded in 96 well plate and treated with samples or DMSO.• After 24 hr. treatment, 100 microlittre of caspase 3/7 reagent were added to each well, mixed and incubated for 1 hr at RT.• Luninescence was measured.• Caspase 3/7 activity was expressed as percentage of untreated cells.
• Sample 1 shows better caspase 3/7 activity at IC 50 then 2*IC 50 value.• Sample 2 did not show caspase activation, suggesting that caspase activation might not be the main pathway for apoptosis induction by 2 acetylfuro 1,4- napthoquinone.
Angiogenesis Test• Quail eggs were purchased.• Test compounds were diluted in 0.1 % DMSO.• 2% Agarose was prepared and mixed in 1:10 ratioof sampele.• Pellet were formed and used for test.• Incubation of eggs at 38 degree C and 80% RH for 70 hr.• After that eggs were opened.• Embryo is transferred in Petri dish.• Placed this in an incubator for 2 hr. at 38 degree C to acclimatize to new ambience.• Test substances were placed on chorioallantropic membrane.• Incubate it for 24 hr.• Imaging of vascularized egg by using digital camera.• Quantitative analysis was performed by software.
• The two compounds showed 65.8 % and 59.6 % inhibition of growth of blood capillaries on embryo membrane in this test.• Suggesting that negative effect on tumor promotion in vivo can be expected.
Discussion• Anticancer activity of 2 acetylfuro 1,4- napthoquinone is being reported first time while the cytotoxicity of Xanthon V1 was reported on HeLa cell lines.• This study confirms the cytotoxic potency of Xanthon V1 on large no. of cancer cell lines.• This work shows the anticancer potency of Santhon V1 and 2 acetylfuro 1,4- napthoquinone .• The most sensitive cell line for Both the samples were Colo-30, HeLa and Caski with IC 50 value closer to that of doxorubicin.• Cytotoxixity of 2 acetylfuro 1,4- napthoquinone was being reported first time.
• For medicle purpose these two compounds can be considered as important.• And the work bring supportive data for future investigations that will lead to their use in cancer chemotherapy.