Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), isolated from Plumbago zeylanica, inhibits ultraviolet radiation-induced development of squamous cell carcinomas

Abstract

Plumbagin (PL) (5-hydroxy-2-methyl-1,4-napthoquinone), a medicinal plant-derived naphthoquinone, was isolated from the roots of the Plumbago zeylanica L. (also known as Chitrak). The roots of P. zeylanica L. have been used in Indian medicine for >2500 years as an anti-atherogenic, cardiotonic, hepatoprotective and neuroprotective agent. We present here that topical application of non-toxic doses (100-500 nmol) of PL to skin elicits dose-dependent inhibition of ultraviolet radiation (UVR)-induced development of squamous cell carcinomas (SCC). In this experiment, FVB/N mice were exposed to UVR (2 kJ/m(2)) three times weekly from a bank of six Kodacel-filtered FS40 sunlamps (∼ 60% UVB and 40% UVA). Carcinoma incidence in mice treated with vehicle, 100, 200 or 500 nmol PL, at 44 weeks post-UVR, were 86, 80 (P = 0.67), 53 (P = 0.12) and 7% (P = 0.0075), respectively. Both vehicle and PL-treated mice gained weight and did not exhibit any signs of toxicity during the entire period of the experiment. Molecular mechanisms associated with inhibition of UVR-induced development of SCC involved induction of apoptosis and inhibition of cell proliferation. Specific findings are that PL treatment (i) inhibited UVR-induced DNA binding of activating protein-1, nuclear factor-kappaB, Stat3 transcription factors and Stat3-regulated molecules (cdc25A and Survivin); (ii) inhibited protein levels of pERK1/2, PI3K85, pAKTSer473, Bcl(2), BclxL, proliferating cell nuclear antigen and cell cycle inhibitory proteins p27 and p21 and (iii) increased UVR-induced Fas-associated death domain expression, poly (ADP-ribose) polymerase protein cleavage and Bax/Bcl(2) ratio. Taken together, our findings suggest that PL may be a novel agent for the prevention of skin cancer.

Fig. 1.

PL treatment inhibits UVR-induced development of SCC. Female FVB/N mice, at 8 weeks of age, were shaved 3 days before the start of UVR exposure and then were exposed to UVR (2 kJ/m²) three times weekly from a bank of six Kodacel-filtered FS40 sunlamps. There were 14 mice per group. Carcinomas were recorded as downward invading lesions, which were confirmed histologically. The carcinoma data are expressed as a percentage of the effectual total. Carcinoma incidence was analyzed using Cox proportional hazards model. (A) Comparison of carcinoma multiplicity and SCC incidence at 42 weeks. (B) Percentage of SCC incidence in vehicle and PL-treated mice at various weeks post-UVR exposure. P values are shown in figures (A and B) comparing vehicle and PL-treated mice. (C) Hematoxylin and eosin staining of uninvolved skin and SCC from vehicle and PL-treated animals. (D) Mouse body weight of vehicle and PL-treated animals at various weeks post-UVR exposure.

Fig. 2.

PL treatment inhibits Stat3, NF-κB and AP-1 DNA binding and expression of phosphorylated Stat3Ser727. Female FVB/N mice were shaved 48 h before experimentation. Mice were exposed to a single dose of 2 kJ/m² UVR and then treated with either vehicle (acetone) or 500 nmol of PL and killed 1, 2 and 3 h post-UVR exposure. Skin was excised and epidermal nuclear lysates and whole epidermal tissue lysates were prepared for Electrophoretic mobility shift assay and western blot analysis, respectively. (A) DNA-binding activity of Stat3, NF-κB and AP-1 by Electrophoretic mobility shift assay. (B) Quantitation of DNA-binding activity of Stat3, NF-κB and AP-1. (C) Protein levels of pStat3Ser727, pStat3Tyr705 and total Stat3 in mouse epidermis obtained from vehicle and PL-treated animals after UVR treatments as determined by western blot analysis. Equal loading of protein was confirmed by stripping and re-probing the blots with actin antibody. (D) Quantitation of the western blots for pStat3Ser727 and pStat3Tyr705 are shown in (C). Arbitrary numbers shown in graph are normalized to actin. (E) Protein levels of survivin and cdc25A in vehicle and PL-treated mouse epidermis tissues as determined by western blot analysis. (F) Quantitation of survivin and cdc25A.

Fig. 3.

PL treatment modulates UVR-induced apoptotic proteins [poly (ADP-ribose) polymerase protein, Bax, Bcl2 and Bcl-xL]. Female FVB/N mice were shaved 48 h before experimentation. Mice are exposed to a single dose of 2 kJ/m² UVR and then treated with either vehicle or PL. Mice were killed 1, 2 and 3 h post-UVR exposure. (A) The epidermal lysate (25 μg protein) was subjected for western blot analysis for indicated proteins. Equal loading of protein was confirmed by stripping and re-probing the blots with actin antibody. (B) Quantitation of the western blots of indicated proteins in (A).

Fig. 4.

Effects of PL on chronic UVR-induced expression of pro-apoptotic and pro-survival proteins. Female FVB/N mice were shaved 48 h before experiment. Mice were exposed to 2 kJ/m² UVR thrice weekly. Vehicle (acetone) or PL was applied 15 min after each UVR treatment for 4 weeks. Mice were killed 6 h after final UVR exposure. The epidermal lysates were prepared and 25 μg proteins were subjected for western blot analysis. Equal loading was confirmed by stripping and re-probing the blots with actin antibody. (A) Dose-dependent effect of PL on protein levels of pAKTSer473, PI3K85, pMEK1/2, MEK1/2, pERK1/2 and ERK1/2. (B) Quantitation of indicated blots shown in (A). (C) Dose-dependent effect of PL on protein levels of FADD, Bax, Bcl2, Bcl-xL, p27 and p21. (D) Quantitation of indicated blots shown in (C). (E) Representative pictures of immunohistochemistry of proliferating cell nuclear antigen (PCNA) in mouse skin obtained from vehicle and PL-treated animals. (F) Bar graph illustrating percentage of PCNA-positive cells in mouse epidermis of vehicle and PL-treated animals. Each value is the percent mean ± standard error of the mean of PCNA-positive cells counted from 10 random areas from each mouse skin.