Plumbagin, a medicinal plant (Plumbago zeylanica)-derived 1,4-naphthoquinone, inhibits growth and metastasis of human prostate cancer PC-3M-luciferase cells in an orthotopic xenograft mouse model

Abstract

We present here first time that Plumbagin (PL), a medicinal plant-derived 1,4-naphthoquinone, inhibits the growth and metastasis of human prostate cancer (PCa) cells in an orthotopic xenograft mouse model. In this study, human PCa PC-3M-luciferase cells (2 × 10(6)) were injected into the prostate of athymic nude mice. Three days post cell implantation, mice were treated with PL (2 mg/kg body wt. i.p. five days in a week) for 8 weeks. Growth and metastasis of PC-3M-luciferase cells was examined weekly by bioluminescence imaging of live mice. PL-treatment significantly (p = 0.0008) inhibited the growth of orthotopic xenograft tumors. Results demonstrated a significant inhibition of metastasis into liver (p = 0.037), but inhibition of metastasis into the lungs (p = 0.60) and lymph nodes (p = 0.27) was not observed to be significant. These results were further confirmed by histopathology of these organs. Results of histopathology demonstrated a significant inhibition of metastasis into lymph nodes (p = 0.034) and lungs (p = 0.028), and a trend to significance in liver (p = 0.075). None of the mice in the PL-treatment group showed PCa metastasis into the liver, but these mice had small metastasis foci into the lymph nodes and lungs. However, control mice had large metastatic foci into the lymph nodes, lungs, and liver. PL-caused inhibition of the growth and metastasis of PC-3M cells accompanies inhibition of the expression of: 1) PKCε, pStat3Tyr705, and pStat3Ser727, 2) Stat3 downstream target genes (survivin and Bcl(xL)), 3) proliferative markers Ki-67 and PCNA, 4) metastatic marker MMP9, MMP2, and uPA, and 5) angiogenesis markers CD31 and VEGF. Taken together, these results suggest that PL inhibits tumor growth and metastasis of human PCa PC3-M-luciferase cells, which could be used as a therapeutic agent for the prevention and treatment of human PCa.

Figure 1

Effects of PL‐treatment on PC‐3M‐luciferase cell‐derived orthotopic xenograft tumors in athymic nude mice. A and B: Luciferase labeled PC‐3M cells (2 × 106) were orthotopically implanted in athymic nude mice. PL (2 mg/kg body weight) and vehicle treatment was started 3 days post‐implantation. There were eight mice per group. Bioluminescence imaging of live mice was performed at the indicated weeks as described in the Methods section. Shown are two representative images of the anesthetized mice from control (Ai) and PL‐treated (Aii) groups. All images were displayed at the same scale. B. The bioluminescence values (photons/sec/cm2/sr) of the prostate region were quantified for each group of mice and mean values ± SE are plotted over time. C. Mice from both groups were sacrificed at 8 week, their prostates were excised and imaged immediately. Bar graph represents the bioluminescence values (photons/sec/cm2/sr) of the excised prostates from control and PL‐treated mice. D. Bar graph represents prostate tumor weights of control and PL‐treated mice. Each value in the graph is the mean ± SE from eight mice. *p < 0.05 was considered as significant.

Figure 2

Effect of PL‐treatment on metastatic growth of PC‐3M‐luciferase cell‐derived tumors in athymic nude mice. At 8 week, livers, lungs, lymph nodes and bone from both vehicle and PL‐treated mice were excised and imaged. A: Representative bioluminescence images of excised livers (Ai–ii), lungs (Aiii–iv), lymph nodes (Av–vi), and bone (Avii–viii) from vehicle (n = 8) and PL‐treated (n = 8) mice. The bioluminescence images of the excised liver, lungs (B) and lymph nodes (C) and bone were quantified for each group of mice. Each value in the graph (B and C) is the mean ± SE from eight mice.

Figure 3

Histopathology of excised prostate tumors, lungs, lymph nodes and liver of control and PL‐treated mice. Ai and Bi: Representative photographs of H&E staining of excised prostates from vehicle treated (Ai) and PL‐treated (Bi) mice at 8 week. T represents xenograft tumors arising from prostate, while green arrows denote to residual mouse prostate glands. Representative H&E staining pictures illustrating distant PCa metastasis into lungs (Aii) and lymph nodes (Aiii) of control mice. Multiple metastatic tumor foci were observed in these tissues as indicated by red arrows. PL‐treated mice showed fewer tumor foci as shown in representative pictures of lungs (Bii), and lymph nodes (Biii), of PL‐treated mice. Representative H&E staining pictures of control (Aiv) and PL‐treated (Biv) animal liver. Control mice showed metastatic tumors in the liver as denoted by MT (Aiv).

Figure 4

Effect of PL‐treatment on PCa metastasis into lungs, lymph nodes and liver. Histopathological evaluation of distant metastasis into lungs (A), lymph nodes (B) and liver (C). 100% stacked column graphs show the percent of single scattered, small metastatic cancer cells and multiple metastastic foci into the lungs (A), lymph nodes (B), and liver (C) of indicated groups.

Figure 5

Effect of PL‐treatment on the expression of PKCϵ, Stat3, MMP2, MMP9, survivin and BclxL. The expression level of the indicated protein was determined by Western blot analysis at eight week from the excised orthotopic xenograft tumor tissues of control and PL‐treated mice. Each lane in the blot represents an individual mouse prostate tumor sample. Equal loading of protein was determined by stripping and re‐probing the blots with actin antibody. Ai: Protein levels of PKCϵ, pStat3Ser727, pStat3Tyr705, and total Stat3. Bi: Protein levels of MMP2, MMP9, survivin and BclxlAii–Bii: Quantification of Western blots of Ai and Bi respectively. Blots were quantitated by densitometric analysis using TotalLab Nonlinear Dynamic Image analysis software.

Figure 6

Effect of PL‐treatment on the expression of VEGF, CD31, uPA, iNOS and cell proliferation markers (Ki‐67 and PCNA). A. Representative immunofluorescent images of VEGF expression in control (Ai) and PL‐treated (Aii) mice excised xenograft tumor tissues. Yellow arrows indicate expression of VEGF. B. Representative immunofluorescent images of CD31 expression in control (Bi) and PL‐treated (Bii) excised xenograft PCa tumor tissues. Red arrows indicate expression of CD31. C. Representative images of immunohistochemistry analysis of CD31 expression in control (Ci) and PL‐treated (Cii) mice excised xenograft tumor tissues. Black arrows indicate expression of CD31 expression. D. Representative images of immunohistochemistry of E‐Cadherin expression in control (Di) and PL‐treated (Dii) mice excised xenograft PCa tumor tissues. E. uPA and iNOS expression in excised xenograft tumor tissues of individual mouse samples of indicated groups as determined by Western blot analysis (Ei). Equal loading of protein was determined by stripping and re‐probing the blot with actin antibody (Ei). Quantification of Western blots by densitometric analysis using TotalLab Nonlinear Dynamic Image analysis software (Eii). F–G. Representative images of immunohistochemistry of PCNA (Fi–ii) and Ki‐67 (Gi–ii) expression in excised xenograft tumor tissues of control and PL‐treated mice.