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

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.

the molecules involved in angiogenesis and metastasis including MMPs and VEGF were studied after treating the cells with DW-F5 for 12, 24 and 48h respectively.
Total protein isolated from cells after indicated treatments were subjected to Western blotting as described earlier 1 . Briefly, 60mg of whole cell protein was resolved on a 10-15% polyacrylamide gel, transferred to a PVDF membrane, incubated with the corresponding antibody and was detected by ECL (Millipore, Billerica, MA, USA).

DNA Fragmentation Assay
For assaying DNA fragmentation, cells were treated with DW-F5 for 48h, scraped into cold PBS and pelleted by centrifugation at 10,000 rpm for 2min. Pellet was dissolved in 400µl of lysis solution (100mM NaCl, 5 mM EDTA, 10 mM Tris-HCl pH 8.0, 0.5% SDS) containing 0.5 mg/ml proteinase-K, followed by overnight incubation at 37°C. After incubation, 100µl of 5M NaCl was added and kept at 4°C for 12h followed by centrifugation at 13,000 rpm for 15 min. and DNA in the supernatant was extracted with 500µl of phenol: chloroform: isoamyl alcohol solution (25:24:1) followed by chloroform: isoamyl alcohol (24:1) treatment.
DNA was precipitated with 1ml of ice-cold absolute ethanol, incubated at -70°C for 30min, centrifuged at 15,000 rpm for 5min, and air-dried. The pellet was dissolved in 20µl distilled water and incubated at 37°C for 30 min after adding 0.5µl of 10mg/ml RNase A. Equal concentrations of DNA were resolved on 2% agarose gel containing ethidium bromide. The bands were visualized under ultraviolet light and photographed using Flour S multiImager (Bio-Rad).

Clonogenic assay
Clonogenic assay was done as mentioned previously 2 . Briefly, 1×10 4 cells in six well plates were treated with DW-F5 for 72h and were replaced with fresh medium and incubated for 1 week. The clones developed were fixed in gluteraldehyde and stained using crystal violet.
The clones were counted and compared with the control. Colony containing more than four cells was counted as one clone.
Preparation of nuclear extract and EMSA: EMSA was performed to evaluate DNAbinding activity of NF-κB as described earlier 2 . In brief, 10 μg of nuclear proteins was incubated for 30 min with 32 P-end-labeled double-stranded NF-ĸB oligonucleotide (5' TTGTTACAAGGGACTTTCC GCTG GGGACTTTCC AGGGAG GCGTGG-3') at 37 o C and the DNA protein complex was resolved in 6.6% non-denaturing polyacrylamide gel, which was dried and visualized by Phosphor Imager (Personal Molecular Imager FX, Bio-Rad).

In vivo studies
All in vivo experiments were approved by the Institute's animal ethical committee (IAEC).

Liposomal encapsulation of DW-F5 fraction
DW-F5 was encapsulated into the uni-lamellar liposome formulation containing phosphatidyl choline and cholesterol as per the method described earlier 4 . Briefly, 5 mg DW-F5, 45 mg phosphatidyl choline and 5.8 mg cholesterol were dissolved in 3:1 mixture of chloroform and methanol (100 ml). After dissolving completely, the solvent was removed using a vacuum rotary evaporator and the residue was suspended in sterile PBS. This solution was sonicated to ensure homogeneity and stored at 4 o C. This preparation was administered to experimental animals at a dose of 120 mg/kg body weight. The control animals were injected with empty liposome vehicle. The drug treatment was continued for a period of four weeks after which the mice were euthanized in CO 2 chamber.

Orthotopic xenograft model
The xenograft studies to evaluate the efficacy of DW-F5 against human skin cancer [melanoma] were carried out in NOD-SCID (NOD.CB17-Prkdc scid /J) mice. Male NOD SCID mice of six to eight weeks old were used in this study and were maintained in animal research facility of our institute. In the standard cage conditions (temperature between 19-25°C, relative humidity 30-70% and illumination cycle set to 12h light and 12h dark), animals were housed in the groups of 4 mice for experiments. Autoclaved rice husk was used as bedding material and autoclaved rodent feed and water was given to animals ad libitum. includes animals receiving the intradermal mode of drug administration and finally Group 3 and 6 [6A and 6B] includes animals receiving the intraperitoneal mode of drug administration. The tumour growth was monitored every two days and tumour volume was measured every week. Throughout the study, all cages were checked every day at regular intervals for any dead or moribund animals, in order to carry out immediate necropsy. As the study could not find any measurable tumour in the day 1 experiment groups [Group 5 and 6] after the completion of the experiment, a set of animals from each groups were again kept under observation [Group 5B and 6B] for two more weeks along with one set of their untreated animals [Group 4B].

Toxicological analysis
Animals were euthanized after the experimental period using CO 2 . Liver tissues of the sacrificed animals was collected and fixed in 4% paraformaldehyde and preserved in 30% sucrose. Tissue Sections obtained from such samples were stained with haematoxylin and eosin 5 . Liver Function Test [LFT] from the serum samples of animas was conducted to analyse the serum levels of various parameters such as total protein, albumin, globulin (all in g/dL) and bilirubin [Total and direct] (mg/dL). The activity of serum alkaline phosphatase (ALP), serum glutamate oxaloacetic transaminase (GOT) and glutamate pyruvic transaminase (GPT) were also estimated (DDRC Laboratories, Trivandrum) and compared with that of normal controls (http://en.aml-vet.com/animal-species/mouse/).

Sample collection and preparation of tissue cryosections
At the termination of the experiment, all the animals were sacrificed using CO 2 . Tumour from both untreated and DW-F5 treated animals were excised, washed in ice-cold PBS and photographed. The radii of tumour in two different planes were measured using a vernier calliper and the volume was calculated as (length X width 2 )/2.

Histology and immunohistochemistry
For histopatholgical examination, the tissue sections were kept in room temperature for 1h, and subsequently passaged twice through PBS, then through distilled water for 5 min each and stained with hematoxylin for 30 min. Excess stain was washed off and the slides were dipped in differentiation solution (3 sec), tap water (10 min) and 70% Isopropyl alcohol (5 min) before counter stained with eosin solution for 1 min. The sections were kept in 100% Isopropyl alcohol for 2 min twice, cleared in xylene and mounted in DPX mountant. Stained sections were observed under a light microscope and photographed.
Immunolocalization of specific proteins in the tissue sections was done using the Super Sensitive Polymer-HRP IHC Detection System (Biogenex, USA). The tissue sections were kept in room temperature for 1h, and subsequently passaged twice through PBS and then through distilled water for 5min each and subjected to heat-induced antigen retrieval in citrate buffer. Nonspecific antibody binding sites on tissue sections and endogenous peroxidase activity were blocked by appropriate reagents supplied with the kit. The primary antibody diluted in TBST was added to the tissue sections. After incubating for 2h at room temperature, the unbound antibody was washed off with PBS-T (Phosphate Buffered Saline with 0.1% Tween-20). The sections were then covered with secondary antibody provided in the kit and incubated for 20min at room temperature followed by rinsing it in PBS-T.
Immunostaining was visualized using diaminobenzidine chromogen, counterstained with Mayer's hematoxylin and the sections were mounted using SuperMount ® mounting medium.
Photomicrographs were captured using a Nikon Eclipse microscope equipped with Image-Pro Plus software.
Fractions having same number of spots (Fr.22,23,24 & 25 and Fr.26,27,28,29 & 30) with similar Rf values on TLC plate were pooled. The pooled fractions were numbered (Fr.5& Fr.6) and were tested for cytotoxic activity. Among these, fraction number five (Fr.5) obtained from third step chromatography showed a single spot in TLC profile. This pure compound was subjected to various spectroscopic techniques for elucidation of the structure. Ruby, A., Kuttan, G., Dinesh Babu, K., Rajasekharan, K. & Kuttan, R. Anti-tumour and antioxidant activity of natural curcuminoids. Cancer. Lett. 94, 79-83 (1995).  Response: Figure 1h is now changed to Figure 2i and 1c-1g is changed to 2c-2h. We did not get any fragmentation in the control sample, except a small smear, which appeared when we tried to make the fragments in the treated wells clearer. Moreover, we have also conducted DAPI staining, Annexin V/PI staining and Annexin PI/ FACS analyses to confirm induction of apoptosis by DW-F5 (Please see Fig 2a & b and Supplimentary Fig 5a). The results have been discussed briefly in the modified version.

Comment [JA5]: Reviewer 3
Comment 1 : The statistic analysis was missed in many quantification data. For figure 1b, what ** and *** mean? No description in the legends. How authors compared and determined the significance of difference among different treatments in time-course and dose dependent manners were not stated (Figure 3b, 3e-3j, Figure 4a and 4b).
Response: Figure

Comment [JA7]: Reviewer 3
Comment 2: The authors used several skin cancer cell lines and finally found that only A375 cells responded apparently to DW-F5. It will be of interest to compare the signaling molecules in figure 2 between/among these cells lines (at least pick one) after DW-F5 treatment.