Anti-melanoma and UV-B protective effect of microbial pigment produced by marine Pseudomonas aeruginosa GS-33

Abstract Bioactivity of a microbial pigment, extracted from fermented broth of culture marine Pseudomonas aeruginosa was screened for anticancer activity against human skin melanoma cell line SK-MEL-2. Upon characterisation, the pigment was confirmed as Phenazine-1-carboxylic acid (PCA). The PCA was found effective against SK-MEL-2 cell line at low concentration (GI50 value <10 μg/mL). Reduced cell density and cell shrinkage with typical morphological changes such as rounding of cells with loss/breaking of cell membrane were seen in SK-MEL-2 cells treated with PCA and Adriamycin. The pigment exhibited UV-B protecting activity as calculated by in vitro spectrophotometric assay and potentiated sun protection factor of commercial sunscreen lotion. Moreover, the pigment was non-toxic up to concentration of 100 ppm as assessed erythrocyte haemolysis assay. These results suggest that microbial pigment PCA could be effective and promising in the treatment as well as prevention of melanoma skin cancers.

. Excessive exposure to solar ultra violet (UV) radiations mainly UV-B (280-320 nm) radiation is the main reason for skin cancer (Kumar et al. 2015). The response of chemotherapy in patients with metastasis is <15% which is unfavourable (Kumar et al. 2012). Therefore, it is necessary to develop alternative strategies employing novel approaches for the prevention and treatment of skin cancer.
Recently, there has been increasing interest in anticancer activity of several naturally occurring microbial pigments such as prodigiosin, violecin, melanin, astaxanthin carotenoid, beta-carotene and phenazine (Cimmino et al. 2012;Venil et al. 2013). Phenazines are bacterial pigments involved in quorum sensing mechanism of Pseudomonas aeruginosa (Pejin et al. 2014). Among naturally occurring phenazines, till date; no report exists about anticancer potential of Phenazine-1-carboxylic acid (PCA), which is one of the prominent secondary metabolite of pseudomonads (Rane et al. 2007). The present studies state potential role of PCA in the prevention as well as treatment of melanoma skin cancer through its UV-B protecting potential and anticancer activity against skin melanoma cell line.
In the present studies, production, purification and characterisation of PCA from marine P. aeruginosa (GS-33) was achieved. The anticancer potential of PCA against human skin melanoma cell line SK-MEL-2 was evaluated. Subsequently, its role in enhancement of sun protection factor (SPF) of commercial sunscreen was evaluated and its toxicity was studied.

Production purification and characterisation of pigment
P. aeruginosa (GS-33) when grown in pigment production medium for 48 h at 28 °C produced coloured compound which upon extraction and purification of yielded thin needle like lemon yellow coloured crystals ( Figure S1). The characterisation of this pigment by UV-vis ( Figure  S2), FTIR ( Figure S3) revealed that pigment have UV absorption peaks at 251 and 367 nm with all characteristics IR peaks for PCA. The pigment was confirmed as PCA from R f value of 0.5294 ( Figure S4), HPLC retention time of 1.97 min ( Figure S5) and mass 225 [M + H] + ( Figure S6).

Anti-melanoma effect of PCA
Purified PCA (at different concentrations) and standard drug Adriamycin (ADR) were evaluated for anticancer activity by using sulforhodamine B assay against human skin melanoma cell line SK-MEL-2 as shown in Figure 1(A). These results depicted that PCA exhibited potent anticancer activity against SK-MEL-2 cells as evident from its GI50 (growth inhibition of 50%) value of 2.30 μg/mL (Table S2) since GI50 value of ≤10 μg/ml is considered to demonstrate anticancer activity in case of pure compounds (Tabassum et al. 2011). PCA showed dose-dependent inhibition of SK-MEL-2 cells proliferation. The GI50 value of PCA was quite comparable to that of ADR. However, the TGI (drug concentration resulting in total growth inhibition) and LC50 (concentration of drug resulting in a net loss of cells by 50% following treatment) values of PCA were higher than that of ADR against SK-MEL-2 cells (Table S2). Typical morphological changes such as rounding of cells with loss/breaking of cell membrane and cell shrinkage can be seen in SK-MEL-2 cells treated with PCA and ADR as compared to untreated cells which are evidences of anticancer activity as showed in Figure 1(B).

UV-B protective effect of PCA
Considering application of sunscreens as an important strategy to prevent or minimise malignant and premalignant skin lesions, UV-B protective effect of PCA and its role in enhancement of SPF have been evaluated. The SPF of commercial sunscreen lotions determined by this method closely matched with labelled SPF. The claimed SPF of these lotions was 15 and 30 while the SPF values determined after analysis was 14.70 ± 0.36 and 29.24 ± 0.41, respectively (Table S3). The SPF of PCA solution in ethanol at concentration 25, 50 and 100 ppm was 1.43 ± 0.08, 2.55 ± 0.07 and 4.73 ± 0.14, respectively. The addition of PCA (25, 50 and 100 ppm) in the solution of two commercial sunscreens caused synergistic 10 to 30% fold increase in their SPF as show in Table S3. Thus, results proved that sunscreen incorporated with PCA have more UV-B protective effect as compared to sunscreen alone.

Toxicity analysis of PCA by erythrocyte haemolysis assay
The permissible limit of haemolysis set for biocompatibility or toxicity assessment of material or biomaterial by erythrocyte haemolysis assay is 5% (Singhal & Ray 2002). The results of erythrocytes haemolysis by PCA are shown in the Figure S7. An overall very less haemolysis was observed for PCA. Although, haemolysis increased with increasing the concentrations of PCA in the present studies, PCA at concentrations up to 100 ppm showed haemolysis within permissible limit. Hence, PCA is a non-toxic to normal human cells at concentration 100 ppm.

Experimental
Experimental details relating to this article are provided as supplementary material, alongside Tables S1-S3 and Figures S1-S7.

Conclusion
Use of natural products can be effective approach in the treatment as well as prevention of cancer. Very low concentration of phenazine pigment (PCA) extracted from marine P. aeruginosa (GS-33) was found to inhibit growth of human skin melanoma cell line (SK-MEL-2), while UV-B protective effect of PCA pointed its potential application in sunscreens. Moreover, the non-toxic nature of PCA against normal human cells (erythrocyte) suggested the biosafety of this pigment.