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. 2015 Jun 10:5:11107.
doi: 10.1038/srep11107.

DW-F5: A novel formulation against malignant melanoma from Wrightia tinctoria

Jayesh Antony  1 , Minakshi Saikia  1 , V Vinod  1 , Lekshmi R Nath  1 , Mohana Rao Katiki  2 , M S R Murty  2 , Anju Paul  1 , A Shabna  1 , Harsha Chandran  1 , Sophia Margaret Joseph  1 , Kumar S Nishanth  3 , Elizabeth Jayex Panakkal  1 , I V Sriramya  1 , I V Sridivya  1 , Sophia Ran  4 , S Sankar  5 , Easwary Rajan  6 , Ruby John Anto  1
Affiliations

DW-F5: A novel formulation against malignant melanoma from Wrightia tinctoria

Jayesh Antony et al. Sci Rep. .

Erratum in

Abstract

Wrightia tinctoria is a constituent of several ayurvedic preparations against skin disorders including psoriasis and herpes, though not yet has been explored for anticancer potential. Herein, for the first time, we report the significant anticancer properties of a semi-purified fraction, DW-F5, from the dichloromethane extract of W. tinctoria leaves against malignant melanoma. DW-F5 exhibited anti-melanoma activities, preventing metastasis and angiogenesis in NOD-SCID mice, while being non-toxic in vivo. The major pathways in melanoma signaling mediated through BRAF, WNT/β-catenin and Akt-NF-κB converging in MITF-M, the master regulator of melanomagenesis, were inhibited by DW-F5, leading to complete abolition of MITF-M. Purification of DW-F5 led to the isolation of two cytotoxic components, one being tryptanthrin and the other being an unidentified aliphatic fraction. The overall study predicts Wrightia tinctoria as a candidate plant to be further explored for anticancer properties and DW-F5 as a forthcoming drug formulation to be evaluated as a chemotherapeutic agent against malignant melanoma.

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Figures

Figure 1
Figure 1. DW-F5 does not induce pharmacological toxicity.
(a,b) DW-F5 induces significant cytotoxicity in the melanoma cell line, A375, while normal skin fibroblast (FS) and epithelial cells (HEMa-LP) are unaffected as determined by microscopic observation and MTT assay. (c,d) H&E stained liver tissues of mice treated with or without DW-F5 for a period of 7 days and 3 months. (e,f) The serum level of total proteins, albumin and globulin in DW-F5 treated mice for a period of 7 days and 3 months is represented graphically. (g,h) Graphical representation of the serum level of total and direct bilirubin in DW-F5 treated mice for a period of 7 days and 3 months. (i,j) The activity of toxicological markers such as serum alkaline phosphatase (ALP), serum glutamic oxaloacetic transaminase (SGOT) and Serum glutamic pyruvic transaminase (SGPT) in DW-F5 treated mice for a period of 7 days and 3 months is graphically represented. Though there was a statistically significant deviation in the values of some toxicological parameters between the control and treated groups, the varied values were within the normal range. Data represent three independent sets of experiments. The error bars represent ± S.D. ANOVA (for MTT assay) or Student’s t test (for toxicological studies) was used for statistical comparison among different groups. * P ≤ 0.05; ** P ≤ 0.01; # P ≤ 0.001, ns non significant.
Figure 2
Figure 2. DW-F5 induces apoptosis in melanoma cells.
(a) Cells were treated with DW-F5 for 24 h, washed with PBS and stained with annexin V–FITC/propidium iodide mixture, and photomicrographed (40X). (b) Cells were exposed to DW-F5 for 48 h, stained with fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide and subjected to flow cytometry. The population of Annexin/ PI-positive cells in the top right and bottom right quadrants represents the total percentage of apoptotic cells. (c–g) Western blots showing DW-F5-induced caspase activation and PARP cleavage in A375 cells. (h) Western blot showing cleaved PARP in A375 cells after DW-F5 treatment (i) Agarose gel showing the effect of DW-F5 on internucleosomal DNA fragmentation in A375 cells. All experiments were repeated thrice. Statistical significance was analysed using Student’s t test. # P ≤ 0.001.
Figure 3
Figure 3. DW-F5 down-regulates activation of survival signals.
(a) DW-F5 drastically inhibits the BRAF expression in A375 cells. (b) DW-F5 down-regulates the constitutive and PMA-induced phosphorylation of ERK1/2. (c-e) DW-F5 down-regulates Brn-2, β-catenin and MITF-M. (f) DW-F5 down-regulates constitutive and PMA-induced phosphorylation of Akt. (g,h) DW-F5 down-regulates nuclear translocation of NF-кB. (i,j) DW-F5 down-regulates constitutive and PMA-induced up-regulation of COX-2 and Bcl-2. (k,l) DW-F5 down-regulates VEGF and MMPs. The details of treatment modes are provided in SI methods.
Figure 4
Figure 4. DW-F5 inhibits melanoma tumourigenesis in NOD-SCID mice.
(a) Representative images of tumour bearing mice which received DW-F5 treatment next day after tumour implantation and the graphical representation of the tumour volume. (Day 1 group) (b) Representative images of mice bearing tumour which received DW-F5 treatment 15 days after tumour implantation (Day-15 group) and the graphical representation of the tumour volume. (c) Images of H&E stained tumour tissue of control and DW-F5 treated mice. (d) Figure showing the expression of PCNA in tumour tissues of control and DW-F5 treated mice. Data represent three independent sets of experiments. The error bars represent ± S.D. Statistical significance was analysed using ANOVA. * P ≤ 0.05; ** P ≤ 0.01; # P ≤ 0.001.
Figure 5
Figure 5. DW-F5 down-regulates key molecules crucial for melanoma survival.
(a–g) Tumour tissues of mice treated with DW-F5 shows marked reduction in the expression of molecules such as (a) BRAF, (b) p-ERK1/2, (c) Brn-2, (d) β-catenin, (e) MITF-M, (f) p-p65 and (g) Bcl-2 as assessed by IHC.
Figure 6
Figure 6. DW-F5 reduces melanoma metastasis.
(a,b) Graph representing reduction in the metastatic spread of A375-Ren-Luc cells in liver and lungs upon DW-F5 treatment, as assessed by in vitro luciferase assay. (c) Tumour sections of mice treated with DW-F5 shows reduction in the expression of MMP-9 as assessed by IHC. Data represent three independent sets of experiments. The error bars represent ± S.D. Statistical significance was analysed by Student’s t test. # P ≤ 0.001.
Figure 7
Figure 7. DW-F5 reduces angiogenesis in NOD SCID mice.
(a) Images displaying the inhibition of blood vessel formation in and around the DW-F5 treated tumour mass with respect to untreated tumour. (b) Reduction of VEGF expression in tumour tissues of DW-F5 administered mice as assessed by IHC.
Figure 8
Figure 8. Purification of DW-F5 leads to the isolation of Tryptanthrin and another aliphatic fraction, which are cytotoxic to melanoma cells.
(a) Flow chart depicting the purification of DW-F5 (b–e) 1H NMR, 13C NMR, Mass spectra and Melting point analysis of the aromatic fraction of DW-F5 (f) Chemical structure of tryptanthrin. (g) 1H NMR spectrum of the aliphatic fraction of DW-F5.
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