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- Title
Ayurvedic Plant Extract Tinospora Cordifola As A Potential Anti-Cancerous Agent Against Hormone Independent Metastatic Breast Cancer.
- Authors
Singh, Jyotsana; Kumar, Brijesh; Kumar, Nikhil; Marshall, John F.; Konwar, Rituraj
- Abstract
Basis of the study-Biological heterogeneity of metastases and epithelial mesenchymal transition (EMT) in breast cancer are two evil faces responsible for poor patient management. Targeting EMT in the breast cancer till now is very challenging and therapeutic interventions are immediately required. Although there are a number of antimetastatic drugs and chemo-therapeutic agents but they have failed to cure the aggressive metastatic tumors compelling the direction of drug discovery towards a newer approach, where the EMT targeting seems to be a promising approach. In present study we have studied the anti-metastatic potential of Tinospora cordifolia Miers (Menispermaceae), also popular as amrita, gurcha, guduchi or jetwatika. The stem extracts were selected for investigation since it has been documented for its immunomodulatory activity. The wide distribution, perennial nature and dioecy of T. cordifolia have led to critically investigate the seasonal and gender based impact on the activity and the content of bioactive compounds by various groups. Recently, our group has successfully developed a statistical approach based on phytochemical markers for confident prediction of variations in metabolic profile and cytotoxicity against MCF-7, MDA-MB-231, DU-145 and PC-3 cancer cells due to geographical, seasonal and gender difference in T. cordifolia stem. Material and Method- Plant materials and chemicals The extract, in form of CDRI-TCSF (Tinospora cordifolia, stem, female) and CDRI-TCSM (Tinospora cordifolia, stem, male) were provided by SAIF (Sophisticated analytical instrument facilities, CSIR-CDRI) division. Male and female plants bearing flowers and fruits (only female plants) were collected from naturally growing population on the banks of the Gomati River, Lucknow in 2012, 2013 and 2014. The plant samples were identified and deposited in the departmental herbarium of the Botany Division of CSIR-CDRI, Lucknow, India. Certified reference material (CRM) of T. cordifolia stem was purchased from Tulsi Amrit Pvt Limited, Indore, India (Batch No. 10TC 1438). Cell culture, cytotoxicity assay and cell cycle analysis MDA-MB-468 were cultured in DMEM media supplemented with 5ml L-glutamine/ 500ml and MCF-10A-CA1a cells were cultured with DMEM:F12 (1:1), 5ml L-glutamine/ 500ml, chlorea toxin (100ng/ml), EGF (20ng/ml), hydrocorticosone (0.5 μg/ml), insulin (10 μg/ml). The CAF (Cancer associated fibroblast) cells were maintained in DMEM:RPMI (1:1ratio) supplemented with 10% FBS and 2.5% penicillin-streptomycin. The cells were grown in 5% without any antibiotics. The test plant extracts were diluted with DMSO in 10mg/mk stock solution and the MTT protocol were followed as already described. The cytotoxicity is performed at 24h, 48h, 72h and 92h. The cells were maintained in humidified environment of 5% CO2and 370C temperature in incubator. Cell cycle assay The propidium iodide dye was used to assay the cell cycle arrest at 48h. In brief, the 1X105cells were seeded, grown for 24h and treated with different concentrations of the PETC for another 48h. Next the cells were harvested and samples were prepared and flow cytometric analysis was performed. Wound Healing Assay The MDA-MB-468 and MCF-10A-Ca1a cells were used for the wound-healing assay at 48h. In short, 1x105 cells per well were seeded onto 6-well plates and grown until 80% of confluency in respective cell culture media supplemented with 10% FBS. One horizontal and one vertical scratch were created using a 10?l sterile micropipette tip on the confluent cell monolayer in each well to ascertain the area under study. The cells were treated with test plant extract. Cells were washed thrice with PBS, added with test compound in medium supplemented with 5% FBS, and monitored for 48h. Images were captured using phasecontrast microscopy at 0 and 48h after treatment with test compound. Cell migration distance was measured using Image-J software (NIH, USA). The result was drawn from three independent experiments and the image is representation of one of the results. EMT path-scan assay Cells (100 cells/well) were seeded in 4 well-chambered slides and grown till cells acquired complete morphology. The cells were then treated with either with test plant extract or without, for 48h, washed thrice with PBS, fixed with 4% paraformaldehyde for 15 min at RT, followed by blocking as per manufacturer instruction (CST, 7771). The cells were next stained with the antibody cocktail; E-cadherin(Alexa Fluor® 488, Ex(max) (nm) 495, Em(max) (nm)- 519)/Vimentin (Alexa Fluor® 555, Ex(max) (nm) 555, Em(max) (nm)-565)antibody cocktail overnight at 4?C in dark. The cells were washed with PBS and counter probed with 2? antibody; the detection cocktail for 2 h in dark. The slides were removed from the cassette and mounted with Prolong® Gold Antifade Reagent having DAPI for nuclear stain. The images were taken on the confocal microscope. Colony formation assay The in-vitro colony formation potentials of the MDA-468 cells and CA1a were analyzed by anchorage-dependent colonogenic assay. 500 cells were counted and seeded into the six well plates in triplicates. After 48h, the cells were exposed to different concentration of CDRI-TCSF and CDRI-TCSM. The cells were cultured in CO2 incubator at 370C. After the experimental exposure time, the treatment media was removed and the cells were left for 7 more days with serum free media, with the media being changed after every 2 days. After incubation, the colonies were fixed with 100% methanol for overnight at -200C. Next the cells were stained with 0.1% crystal violet dissolved in the methanol (Sigma-Aldrich). The colonies were counted manually with ?50 cells/colony and expressed as percent control. 3D organotypic assay The 3D organotypic assay was performed with MDA-MB-468 and MCF-10A-CA1a cells along with two CAFs, 2276 and 1939. Briefly, the gel was prepared with Matrigel (BD 354243) and collagen Type I (BD 355236). The transwell chamber were coated with 300?l collagen Type I (1:100 dilution in PBS) and incubated at 370C, 1h. Excess collagen wad removed and 120 ?l/well matrigel/collagen was added without any bubble formation. The gel was left for 1-2 h to set followed by addition of media without letting it to be dried out. The cells were trypsinised and counted. Total 100, 000 cells/well cells were required in 200 μl (culture media was same as required to grow the cells under study) of media. For cell mixture, 67, 000 fibroblast (i.e. (100, 000/3)*2) and 33, 000 cancer cells were required. 600?l of same media was added underneath transwell through top slit of transwell. The cells were left to grow for 24 h. Next, after 24h the media was removed and added with fresh 350 ?l culture media (either with test plant extract or without) at the bottom and top surface of the organotypic culture gel was feeded with plain cell culture media without any FBS (either with test plant extract or without). The media is replaced after every 2 day until the termination of the experiment. At the end of the experiment, the transwells were placed in the formalin with 200 ?l above and 600 ?l below the transwell surface inside the chamber dishes, overnight. Next day, formalin was discarded and replaced by 70% ethanol for atleast 10 minutes with 200 ?l above and 600 ?l below the transwell surface inside the chamber dishes. After fixation the gel is embedded in the paraffin and sectioned for the further study. LA-7 induced syngenic mammary tumor model In brief, animal were injected 106 cells/ animal in the mammary fat pad with 10ml syringe in form of cell suspension in the LA-7 culture media of female SD rats. The animals were administered with ciclosporin subcutaneously (80mg/kg animal body weight, dissolved in olive oil, 100 μl with insulin syringe). The animals were left for 10 days for tumor formation and regularly monitored for tumor growth; measured after every 3 day. Tumors were measured with vernier calipers. After attaining a measurable tumor size, test plant extracts were administered in the animals orally at the dose of 75mg/ kg either CDRI-TCM or CDRI-TCF or PTX weight with gamacasia to enhance the bioavailability of treatment in animal body. The control group was vehicle (0.01% DMSO) treated. After 21 days of study the animals were sacrificed by cervical dislocation. The animal body weights were recorded at regular interval with survival index. The weight of vital organs like lung, liver, kidney, heart, spleen and tumor were harvested in saline solution and recorded at the termination of the experiment. The lungs were further studied for any neoplastic lesions with Bouin's solution for overnight. The lesions were counted manually. Primary cell culture The ER/PR-ve breast tumor samples were collected and assayed to evaluate the impact of TCSF and TCSM after 48h of treatment. In brief, the tumor tissues were collected, minced, digested and re-suspended DMEM-high glucose medium. Supernatant was discarded, the pellets were washed in cold PBS and centrifuged again at 750 g for 10min at RT. Pellets were then suspended in PBS and filtered. The cell suspension was then centrifuged at 40 g (1min); only supernatant was transferred into new 50mL tubes and centrifuged at 100 g (2min) obtained the pellet consist of epithelial fraction The entire procedure was done in sterile conditions. The cells were culture at 370 C, 5% CO2 and under humidified condition. Statistical analysis The statistical significance of difference between the experimental groups were determined with one way ANOVA, Tukey multiple tests on GraphPad Prism Version 5.0 software. Two-way ANOVA analysis was used to determine the tumor volume and total tumor burden in the in vivo animal groups. All the experiments were done at-least thrice. Results- We have studied the influence of gender variations of PETC on anti-breast cancerous activity. The aggressive breast cancer cells, MDA-MB-468 and MCF- 10A-CA1a were selected for 2D and 3D studies and female syngenic tumor bearing SD rats were selected as in vivo model. TCSF and TCSM induced significant cytotoxicity to the breast cancer cell and both resulted in the induction of G2/M arrest in highly aggressive MDA-MB-468 cells, conferring the anti-cancerous potentials. The extracts resulted in significant inhibition of cancer cell migration and colony formation in both the cell lines with much higher significant inhibition in MDA-MB-468. The key regulators of EMT, E-cadherin and Vimentin were significantly modulated by both TCSM and TCSF with significant higher activity in the TCSM. Vimentin was down regulated and E-cadherin was found to up regulated. In 3D organotypic model, we observed that the invasive and migratory potential were significantly reduced by TCSM extract as compared to the TCSF. Our in vivo study on syngeneic rat model also validated the results that the TCSM possesses better potential to be developed as therapeutic intervention against breast cancer when compared with the TCSF. We have next studied activity on highly metastatic ER/PR negative patient-derived primary tumor culture where TCSM significantly inhibited the proliferation. However, we have worked on the crude plant extract of T. cordifolia, hence validation of the anti-cancerous property on pure compounds derived from T. cordifolia and their comparison among TCSM and TCSF plant is warranted. Conclusion- 1. First experimental evidence against the anti-cancerous activity of the ayurvedic medicinal plant is affected by regional variation as well as dioecious nature of plant. 2. In vitro results show that both PETC extracts has potent anti-cancerous activity with significantly higher potency in the TCSM. 3. Significantly reduced EMT-MET and metastases in 2D and 3D microenvironment. 4. Studies on syngeneic SD rat mammary tumor model showed significant tumor regression in TCSF and TCSM with much higher reduction in TCSM. 5. We are first to report that the male plant possesses better anti-tumor potential and could be developed as therapeutic intervention against metastatic and aggressive breast cancer.
- Subjects
LUCKNOW (India); INDORE (India); METASTATIC breast cancer; PLANT extracts; CELL migration inhibition; CANCER cell migration; CELL analysis; CULTURE media (Biology); GLUTAMINE
- Publication
Journal of Cancer Research & Therapeutics, 2017, Vol 13, pS97
- ISSN
0973-1482
- Publication type
Article