Plumbagin, a plant derived natural agent inhibits the growth of pancreatic cancer cells in in vitro and in vivo via targeting EGFR, Stat3 and NF-κB signaling pathways

Abstract

Pancreatic cancer (PC) is the most aggressive malignant disease, ranks as the fourth most leading cause of cancer-related death among men and women in the United States. We present here that plumbagin (PL), a quinoid constituent isolated from the roots of the medicinal plant Plumbago zeylanica L, inhibits the growth of PC cells both in vitro and in vivo model systems. PL treatment induces apoptosis and inhibits cell viability of PC cells (PANC1, BxPC3 and ASPC1). In addition, i.p. administration of PL (2 mg/kg body weight, 5 days a week) in severe combined immunodeficiency (SCID) mice beginning 3 days after ectopic implantation of PANC1 cells resulted in a significant (P < 0.01) inhibition of both tumor weight and volume. PL treatment inhibited (1) constitutive expression of epidermal growth factor receptor (EGFR), pStat3Tyr705 and pStat3Ser727, (2) DNA binding of Stat3 and (3) physical interaction of EGFR with Stat3, in both cultured PANC1 cells and their xenograft tumors. PL treatment also inhibited phosphorylation and DNA-binding activity of NF-κB in both cultured PC cells (PANC1 and ASPC1) and in PANC1 cells xenograft tumors. Downstream target genes (cyclin D1, MMP9 and Survivin) of Stat3 and NF-κB were similarly inhibited. These results suggest that PL may be used as a novel therapeutic agent against human PC. Published 2012 Wiley-Liss, Inc. This article is a US Government work, and, as such, is in the public domain in the United States of America.

Figure 1. PL inhibits cell viability, cell invasion and induces apoptosis in PC cells

A. Effect of PL on viability of PANC1 (Ai), BxPC3 (Aii) and ASPC1 (Aiii) cells as determined by MTT assay. The values are represented as percent viable cells as compared to the vehicle treated cells. Each value is the mean± SE of each group. B. PL induces apoptosis in PANC1 cells as observed by Annexin V staining (Bi–Biv), and the images were captured with a fluorescent microscope. GF (green field) represents Annexin V staining while BF (bright field) pictures represent the total number of cells from the same field. C. PL induces PARP cleavage as determined by Western blot analysis. D. Effect of PL on cell invasion of PANC1 cells. Representative photographs of invaded cells of untreated (Di) and PL treated (Dii–iv) cells. E. Number of invading cells was determined by colorimetric measurement at 490 nM according to the manufacturer’s instructions. Each indicated group value in the graph is the mean± S.E from 3 separate wells.

Figure 2. PL inhibits both Stat3 activation and dissociates Stat3-interaction with EGFR

A. PL inhibits protein level of EGFR, pStat3Tyr705, pStat3Ser727 in PANC1 (Ai) BxPC3 (Aii) and ASPC1 (Aiii) cells as determined by Western blot analysis. Equal loading of protein was determined by stripping and reprobing the blots with actin antibody. Values in arbitrary number (A.N) shown above the immunoblots represent densitometer quantitation of band normalized to actin. B. Dose (Bi) and time-dependent (Bii) effect of PL on Stat3 DNA binding activity in PANC1 cells as determined by EMSA. In the blot (Bi) Lane 6 represents the mutant consensus sequence of Stat3. C. Effect of PL on physical association of EGFR with Stat3 in PANC1 cells as determined by immunoprecipitation and blotting experiment. D. Effect of PL on co-localization of EGFR and Stat3 in PANC1 cells as determined by double immunofluorescence. White arrows indicate the expression of EGFR, yellow arrows indicate expression of Stat3, and green arrows denote co-localization of EGFR with Stat3.

Figure 3. PL inhibits activation of NF-κB in PC cells

A. Expression level of pNF-κB/p65Ser536 and total NF-κB/p65 in PANC1 (Ai) BxPC3 (Aii) and ASPC1 (Aiii) cells as determined by Western blot analysis. B. EMSA assays represent dose and time-dependent effect of PL on DNA binding activity of NF-κB in PANC1 (Bi–Bii) and BxPC3 (Biii–Biv) cells. Lane 6 of Bi and Biii EMSA blot represent mutant consensus sequence of NF-κB. C. Localization of NF-κB/p65 in PANC1 cells as determined by immunocytochemistry. Representing pictures illustrating DAPI (Ci), NF-κB/p65 (Cii) staining and merged images of DAPI and NF-κB/p65 (Ciii) in control (upper panel) and PL treated cells (Lower panel). D. Effect of PL on protein levels of IKKγ, pIkBα, and total IkBα in PANC1 (Di) and ASPC1 (Dii) cells as determined by Western blot analysis. (E) Expression levels of Cdc25A, Cyclin D1, MMP9, and survivin as determined by Western blot analysis.

Figure 4. PL-treatment inhibits the growth of ectopically xenograft PANC1 cells tumors (A–B) in SCID mice and decreases serum IL-6 levels (D)

A. Representative photographs of the SCID mice bearing PANC1 xenograft and excised tumors of control (Ai) and PL-treated group (Aii) after 66 days post-implantation. B. Average tumor volume of vehicle or PL-treated animals was plotted over days. Each value on the graph represents the mean and S.E of 16 tumors. C. Effect of PL on tumor weight of control and PL-treated animals after 66 days post-implantation of PC cells. Values in bar graph represent mean±SE of 16 tumors of each group. Asterisks represent level of significance (P<0.01). D. Effect of PL on serum IL-6 levels as determined by ELISA assay. Values in bar graph represent mean±SE from 8 animals.

Figure 5. PL treatment inhibits EGFR, Stat3 and NF-κB/p65 expression in PC cell xenograft tumors

A. Effect of PL on the expression levels of EGFR, pStat3Tyr705, pStat3Ser727 in PC cells xenograft tumors as determined by Western blot analysis. Expression of EGFR, pStat3Ser727, pStat3Tyr705 and total Stat3 (Ai). Each lane represents pooled samples of two animal xenograft tumors. Lane 1, 2, and 3 denote control, whereas lane 4, 5, and 6 represent PL-treated group. Bar graph values represent the arbitrary number normalized with GAPDH (Aii). B–C. Effect of PL on physical interaction of Stat3 with EGFR as determined by reciprocal immunoprecipitation and Western blot analysis. BP in Fig. C denotes to blocking peptide of EGFR. D. Effect of PL on co-localization of EGFR and Stat3 as determined by double immunofluorescence. Representative images of DAPI staining (Di), EGFR staining (Dii), Stat3 staining (Diii) and co-localization of EGFR and Stat3 (Div) of control (upper panel) or PL-treated (lower panel) xenograft tumors. E. Effect of PL on activation of NF-κB in PC cells xenograft tumors. Ei. Western blot analysis of pNF-κB/p65Ser536 in control and PL-treated xenograft tumors. Bar graph values represent the arbitrary number normalized with actin (Eii).

Figure 6. PL-treatment inhibits markers of cell proliferation and metastasis in PC cell xenograft tumors

Expression levels of PCNA (Ai–Aii upper panel), ki67 (Ai–Aii middle panel), and VEGF (Ai–Aii lower panel) in control and PL-treated xenograft tumors as determined by immunohistochemistry. Aiii: Negative control by using IgG. Bi–Bii: Bar graphs represent quantification of PCNA nuclear staining in control and PL-treated xenograft tumors. Student t-test was performed to analyze nuclear staining difference (P<0.05).