Novel quinazoline-based compounds impair prostate tumorigenesis by targeting tumor vascularity

Abstract

Previous evidence showed the ability of the quinazoline-based alpha(1)-adrenoreceptor antagonist doxazosin to suppress prostate tumor growth via apoptosis. In this study, we carried out structural optimization of the chemical nucleus of doxazosin and a subsequent structure-function analysis toward the development of a novel class of apoptosis-inducing and angiogenesis-targeting agents. Our lead compound, DZ-50, was effective at reducing endothelial cell viability via a nonapoptotic mechanism. Treatment with DZ-50 effectively prevented in vitro tube formation and in vivo chorioallantoic membrane vessel development. Confocal microscopy revealed a significantly reduced ability of tumor cells to attach to extracellular matrix and migrate through endothelial cells in the presence of DZ-50. In vivo tumorigenicty studies using two androgen-independent human prostate cancer xenografts, PC-3 and DU-145, showed that DZ-50 treatment leads to significant suppression of tumorigenic growth. Exposure to the drug at the time of tumor cell inoculation led to prevention of prostate cancer initiation. Furthermore, DZ-50 resulted in a reduced formation of prostate-tumor derived metastatic lesions to the lungs in an in vivo spontaneous metastasis assay. Thus, our drug discovery approach led to the development of a class of lead (quinazoline-based) compounds with higher potency than doxazosin in suppressing prostate growth by targeting tissue vascularity. This new class of quinazoline-based compounds provides considerable promise as antitumor drugs for the treatment of advanced prostate cancer.

Figure 1. Effect of novel lead quinazoline-derived compound DZ-50 on human prostate cancer cells

(a) Chemical Structure of DZ-50: The 2,3-difydro-benzo [1,4] dioxane0-carbonyl moitey of doxazisin was replaced with the bi-phenyl aryl sulfonyl substituent; while the methoxy side chains were replaced with isopropyl propoxy functions. (b) Apoptosis induction by novel quinazoline compounds. PC-3 cells were treated (10μM) for 24hrs and apoptosis was measured by Hoechst staining. (c) Apoptosis induction by DZ-3. FACS analysis of PI and BrdU staining was performed on PC-3 cells treated with DZ-3 (10μM) and a negative control, DZ-50 (10μM). (d) Cell death following DZ-50 treatment. Cell death was evaluated in endothelial and epithelial cells lines following a 24hr and 48hr (inset) treatment of DZ-50 (5μM, 10μM, 15μM, 20μM, and 25μM) as described in “Methods”.

Figure 1. Effect of novel lead quinazoline-derived compound DZ-50 on human prostate cancer cells

(a) Chemical Structure of DZ-50: The 2,3-difydro-benzo [1,4] dioxane0-carbonyl moitey of doxazisin was replaced with the bi-phenyl aryl sulfonyl substituent; while the methoxy side chains were replaced with isopropyl propoxy functions. (b) Apoptosis induction by novel quinazoline compounds. PC-3 cells were treated (10μM) for 24hrs and apoptosis was measured by Hoechst staining. (c) Apoptosis induction by DZ-3. FACS analysis of PI and BrdU staining was performed on PC-3 cells treated with DZ-3 (10μM) and a negative control, DZ-50 (10μM). (d) Cell death following DZ-50 treatment. Cell death was evaluated in endothelial and epithelial cells lines following a 24hr and 48hr (inset) treatment of DZ-50 (5μM, 10μM, 15μM, 20μM, and 25μM) as described in “Methods”.

Figure 1. Effect of novel lead quinazoline-derived compound DZ-50 on human prostate cancer cells

(a) Chemical Structure of DZ-50: The 2,3-difydro-benzo [1,4] dioxane0-carbonyl moitey of doxazisin was replaced with the bi-phenyl aryl sulfonyl substituent; while the methoxy side chains were replaced with isopropyl propoxy functions. (b) Apoptosis induction by novel quinazoline compounds. PC-3 cells were treated (10μM) for 24hrs and apoptosis was measured by Hoechst staining. (c) Apoptosis induction by DZ-3. FACS analysis of PI and BrdU staining was performed on PC-3 cells treated with DZ-3 (10μM) and a negative control, DZ-50 (10μM). (d) Cell death following DZ-50 treatment. Cell death was evaluated in endothelial and epithelial cells lines following a 24hr and 48hr (inset) treatment of DZ-50 (5μM, 10μM, 15μM, 20μM, and 25μM) as described in “Methods”.

Figure 1. Effect of novel lead quinazoline-derived compound DZ-50 on human prostate cancer cells

(a) Chemical Structure of DZ-50: The 2,3-difydro-benzo [1,4] dioxane0-carbonyl moitey of doxazisin was replaced with the bi-phenyl aryl sulfonyl substituent; while the methoxy side chains were replaced with isopropyl propoxy functions. (b) Apoptosis induction by novel quinazoline compounds. PC-3 cells were treated (10μM) for 24hrs and apoptosis was measured by Hoechst staining. (c) Apoptosis induction by DZ-3. FACS analysis of PI and BrdU staining was performed on PC-3 cells treated with DZ-3 (10μM) and a negative control, DZ-50 (10μM). (d) Cell death following DZ-50 treatment. Cell death was evaluated in endothelial and epithelial cells lines following a 24hr and 48hr (inset) treatment of DZ-50 (5μM, 10μM, 15μM, 20μM, and 25μM) as described in “Methods”.

Figure 2. DZ-50 prevents cell migration and adhesion to ECM of human prostate tumor epithelial cells and vascular endothelial cells

(a) Wounding assays were performed on endothelial and epithelial cells and the number of migratory cells was quantified (as described in the “Methods”). There was a significant reduction in the migratory capacity detected in the vascular endothelial and tumor epithelial cells analyzed; * P < 0.0001, ** P < 0.001, *** P 0.004. (b, c) DZ-50 prevents prostate tumor epithelial cell attachment to ECM components. The ability of prostate cancer cells PC-3 to adhere to ECM protein components was evaluated after exposure to DZ-50 for 6, 9, and 12hrs at concentrations of 5μM and 10μM. Attached prostate cancer cells were counted on fibronectin or collagen coated culture dishes (mean ± SD.). DZ-50 significantly reduced the ability of PC-3 cells to attach to either fibronectin or collagen. (d, e) DZ-50 prevents prostate cancer epithelial cell adhesion to endothelial cells. Transdendothelial migration assays were performed to assess the ability of PC-3 prostate cancer cells to attach and migrate through a monolayer of HMVEC-L following exposure to DZ-50. (d) PC-3 cells were stained with the lipophilic tracer DiI (red) and were subsequently added to a confluent monolayer of HMVEC-L and exposed to DZ-50 for 3 and 9hrs. DAPI staining identified the nuclei (blue). Epithelial cell adhesion to the endothelial cell monolayer was prevented following 9hrs of exposure to the drug (10μM). Cell viability assays were performed on PC-3 and HMVEC-L cells treated with DZ-50 (10μM). No death was detected within the first 24hrs of treatment, indicating that the effect on transendothelial tumor cell migration was not due to drug-induced loss of cell viability (e).

Figure 2. DZ-50 prevents cell migration and adhesion to ECM of human prostate tumor epithelial cells and vascular endothelial cells

(a) Wounding assays were performed on endothelial and epithelial cells and the number of migratory cells was quantified (as described in the “Methods”). There was a significant reduction in the migratory capacity detected in the vascular endothelial and tumor epithelial cells analyzed; * P < 0.0001, ** P < 0.001, *** P 0.004. (b, c) DZ-50 prevents prostate tumor epithelial cell attachment to ECM components. The ability of prostate cancer cells PC-3 to adhere to ECM protein components was evaluated after exposure to DZ-50 for 6, 9, and 12hrs at concentrations of 5μM and 10μM. Attached prostate cancer cells were counted on fibronectin or collagen coated culture dishes (mean ± SD.). DZ-50 significantly reduced the ability of PC-3 cells to attach to either fibronectin or collagen. (d, e) DZ-50 prevents prostate cancer epithelial cell adhesion to endothelial cells. Transdendothelial migration assays were performed to assess the ability of PC-3 prostate cancer cells to attach and migrate through a monolayer of HMVEC-L following exposure to DZ-50. (d) PC-3 cells were stained with the lipophilic tracer DiI (red) and were subsequently added to a confluent monolayer of HMVEC-L and exposed to DZ-50 for 3 and 9hrs. DAPI staining identified the nuclei (blue). Epithelial cell adhesion to the endothelial cell monolayer was prevented following 9hrs of exposure to the drug (10μM). Cell viability assays were performed on PC-3 and HMVEC-L cells treated with DZ-50 (10μM). No death was detected within the first 24hrs of treatment, indicating that the effect on transendothelial tumor cell migration was not due to drug-induced loss of cell viability (e).

Figure 2. DZ-50 prevents cell migration and adhesion to ECM of human prostate tumor epithelial cells and vascular endothelial cells

(a) Wounding assays were performed on endothelial and epithelial cells and the number of migratory cells was quantified (as described in the “Methods”). There was a significant reduction in the migratory capacity detected in the vascular endothelial and tumor epithelial cells analyzed; * P < 0.0001, ** P < 0.001, *** P 0.004. (b, c) DZ-50 prevents prostate tumor epithelial cell attachment to ECM components. The ability of prostate cancer cells PC-3 to adhere to ECM protein components was evaluated after exposure to DZ-50 for 6, 9, and 12hrs at concentrations of 5μM and 10μM. Attached prostate cancer cells were counted on fibronectin or collagen coated culture dishes (mean ± SD.). DZ-50 significantly reduced the ability of PC-3 cells to attach to either fibronectin or collagen. (d, e) DZ-50 prevents prostate cancer epithelial cell adhesion to endothelial cells. Transdendothelial migration assays were performed to assess the ability of PC-3 prostate cancer cells to attach and migrate through a monolayer of HMVEC-L following exposure to DZ-50. (d) PC-3 cells were stained with the lipophilic tracer DiI (red) and were subsequently added to a confluent monolayer of HMVEC-L and exposed to DZ-50 for 3 and 9hrs. DAPI staining identified the nuclei (blue). Epithelial cell adhesion to the endothelial cell monolayer was prevented following 9hrs of exposure to the drug (10μM). Cell viability assays were performed on PC-3 and HMVEC-L cells treated with DZ-50 (10μM). No death was detected within the first 24hrs of treatment, indicating that the effect on transendothelial tumor cell migration was not due to drug-induced loss of cell viability (e).

Figure 2. DZ-50 prevents cell migration and adhesion to ECM of human prostate tumor epithelial cells and vascular endothelial cells

(a) Wounding assays were performed on endothelial and epithelial cells and the number of migratory cells was quantified (as described in the “Methods”). There was a significant reduction in the migratory capacity detected in the vascular endothelial and tumor epithelial cells analyzed; * P < 0.0001, ** P < 0.001, *** P 0.004. (b, c) DZ-50 prevents prostate tumor epithelial cell attachment to ECM components. The ability of prostate cancer cells PC-3 to adhere to ECM protein components was evaluated after exposure to DZ-50 for 6, 9, and 12hrs at concentrations of 5μM and 10μM. Attached prostate cancer cells were counted on fibronectin or collagen coated culture dishes (mean ± SD.). DZ-50 significantly reduced the ability of PC-3 cells to attach to either fibronectin or collagen. (d, e) DZ-50 prevents prostate cancer epithelial cell adhesion to endothelial cells. Transdendothelial migration assays were performed to assess the ability of PC-3 prostate cancer cells to attach and migrate through a monolayer of HMVEC-L following exposure to DZ-50. (d) PC-3 cells were stained with the lipophilic tracer DiI (red) and were subsequently added to a confluent monolayer of HMVEC-L and exposed to DZ-50 for 3 and 9hrs. DAPI staining identified the nuclei (blue). Epithelial cell adhesion to the endothelial cell monolayer was prevented following 9hrs of exposure to the drug (10μM). Cell viability assays were performed on PC-3 and HMVEC-L cells treated with DZ-50 (10μM). No death was detected within the first 24hrs of treatment, indicating that the effect on transendothelial tumor cell migration was not due to drug-induced loss of cell viability (e).

Figure 2. DZ-50 prevents cell migration and adhesion to ECM of human prostate tumor epithelial cells and vascular endothelial cells

(a) Wounding assays were performed on endothelial and epithelial cells and the number of migratory cells was quantified (as described in the “Methods”). There was a significant reduction in the migratory capacity detected in the vascular endothelial and tumor epithelial cells analyzed; * P < 0.0001, ** P < 0.001, *** P 0.004. (b, c) DZ-50 prevents prostate tumor epithelial cell attachment to ECM components. The ability of prostate cancer cells PC-3 to adhere to ECM protein components was evaluated after exposure to DZ-50 for 6, 9, and 12hrs at concentrations of 5μM and 10μM. Attached prostate cancer cells were counted on fibronectin or collagen coated culture dishes (mean ± SD.). DZ-50 significantly reduced the ability of PC-3 cells to attach to either fibronectin or collagen. (d, e) DZ-50 prevents prostate cancer epithelial cell adhesion to endothelial cells. Transdendothelial migration assays were performed to assess the ability of PC-3 prostate cancer cells to attach and migrate through a monolayer of HMVEC-L following exposure to DZ-50. (d) PC-3 cells were stained with the lipophilic tracer DiI (red) and were subsequently added to a confluent monolayer of HMVEC-L and exposed to DZ-50 for 3 and 9hrs. DAPI staining identified the nuclei (blue). Epithelial cell adhesion to the endothelial cell monolayer was prevented following 9hrs of exposure to the drug (10μM). Cell viability assays were performed on PC-3 and HMVEC-L cells treated with DZ-50 (10μM). No death was detected within the first 24hrs of treatment, indicating that the effect on transendothelial tumor cell migration was not due to drug-induced loss of cell viability (e).

Figure 3. DZ-50 prevents angiogenesis in vitro and in vivo

(a,b) In vitro angiogenesis is blocked following exposure to DZ-50. Endothelial cells were seeded in Matrigel in the presence or absence of either DZ-50 or doxazosin at 10μM concentration and tube formation was visualized and quantified in the presence or absence of VEGF, as described in “Methods”. The control (upper panel) shows HUVEC tube formation with decisive branch points while the DZ-50 severely abrogated branch point formation. Panel b, reveals the quantitative analysis of the data; a significant reduction in tube formation is detected in the presence of DZ-50 compared to controls while the novel quinazoline DZ-10 (no effect on cell viability-negative control) does not change the ability of HUVEC cells to form multi-branched tubular networks. VEGF, cannot reverse the antiangiogenic effect of DZ-50, (c,d) In vivo angiogenesis is blocked by DZ-50. CAM assays were performed in the presence or absence of DZ-50 as described in “Methods” and the number of blood vessels were counted.

Figure 3. DZ-50 prevents angiogenesis in vitro and in vivo

(a,b) In vitro angiogenesis is blocked following exposure to DZ-50. Endothelial cells were seeded in Matrigel in the presence or absence of either DZ-50 or doxazosin at 10μM concentration and tube formation was visualized and quantified in the presence or absence of VEGF, as described in “Methods”. The control (upper panel) shows HUVEC tube formation with decisive branch points while the DZ-50 severely abrogated branch point formation. Panel b, reveals the quantitative analysis of the data; a significant reduction in tube formation is detected in the presence of DZ-50 compared to controls while the novel quinazoline DZ-10 (no effect on cell viability-negative control) does not change the ability of HUVEC cells to form multi-branched tubular networks. VEGF, cannot reverse the antiangiogenic effect of DZ-50, (c,d) In vivo angiogenesis is blocked by DZ-50. CAM assays were performed in the presence or absence of DZ-50 as described in “Methods” and the number of blood vessels were counted.

Figure 3. DZ-50 prevents angiogenesis in vitro and in vivo

(a,b) In vitro angiogenesis is blocked following exposure to DZ-50. Endothelial cells were seeded in Matrigel in the presence or absence of either DZ-50 or doxazosin at 10μM concentration and tube formation was visualized and quantified in the presence or absence of VEGF, as described in “Methods”. The control (upper panel) shows HUVEC tube formation with decisive branch points while the DZ-50 severely abrogated branch point formation. Panel b, reveals the quantitative analysis of the data; a significant reduction in tube formation is detected in the presence of DZ-50 compared to controls while the novel quinazoline DZ-10 (no effect on cell viability-negative control) does not change the ability of HUVEC cells to form multi-branched tubular networks. VEGF, cannot reverse the antiangiogenic effect of DZ-50, (c,d) In vivo angiogenesis is blocked by DZ-50. CAM assays were performed in the presence or absence of DZ-50 as described in “Methods” and the number of blood vessels were counted.

Figure 3. DZ-50 prevents angiogenesis in vitro and in vivo

(a,b) In vitro angiogenesis is blocked following exposure to DZ-50. Endothelial cells were seeded in Matrigel in the presence or absence of either DZ-50 or doxazosin at 10μM concentration and tube formation was visualized and quantified in the presence or absence of VEGF, as described in “Methods”. The control (upper panel) shows HUVEC tube formation with decisive branch points while the DZ-50 severely abrogated branch point formation. Panel b, reveals the quantitative analysis of the data; a significant reduction in tube formation is detected in the presence of DZ-50 compared to controls while the novel quinazoline DZ-10 (no effect on cell viability-negative control) does not change the ability of HUVEC cells to form multi-branched tubular networks. VEGF, cannot reverse the antiangiogenic effect of DZ-50, (c,d) In vivo angiogenesis is blocked by DZ-50. CAM assays were performed in the presence or absence of DZ-50 as described in “Methods” and the number of blood vessels were counted.

Figure 4. DZ-50 targets integrin expression profile in human prostate cancer cells

(a) Comparison of integrin β₁ expression on PC-3 prostate cells following 12hrs exposure of DZ-50 (10μM) or vehicle control (DMSO). (b) Comparison of integrin β₁ expression on DU-145 prostate cells following 12hrs exposure of DZ-50 (10μM) or vehicle control (DMSO).

Figure 5. Suppression of primary tumor growth and prevention of prostate tumor development in human prostate cancer xenograft model by DZ-50

(a, b) Tumor volume of prostate xenografts is reduced following DZ-50 treatment. Following subcutaneous inoculation of nude mice (n = 6 per group) with either PC-3 (panel a) or DU-145 (panel b) human prostate cancer cells, DZ-50 (100mg/kg and 200mg/kg) was administered orally (via oral-gavage) to tumor-bearing-hosts for 14 days (subsequent to palpable tumor formation). Tumor volume was measured daily as described in “Methods”; DZ-50 treatment significantly suppressed prostate tumor volume compared to the vehicle control, P < 0.001. (c) Primary prevention of androgen-independent human prostate cancer by DZ-50. To determine the ability of the new lead drug to prevent prostate cancer development, nude mice were subcutaneously inoculated (n = 6 per group) with PC-3 cells with concurrent exposure (orally) to DZ-50 (200mg/kg) for 2-wks. (d) Prostate cancer xenografts were excised from DZ-50-treated and vehicle control tumor-bearing mice, paraffin-embedded, and tissue sections (6μM) were subjected to immunohistochemical analysis for apoptosis, cell proliferation and tumor vascularity, (panels a and b). The three panels represent TUNEL staining for apoptosis, CD31 immunoreactivity for vascularity, and Ki67 expression for cell proliferation, (Magnification 400x). Quantitative analysis of the results is shown on Table 1.

Figure 5. Suppression of primary tumor growth and prevention of prostate tumor development in human prostate cancer xenograft model by DZ-50

(a, b) Tumor volume of prostate xenografts is reduced following DZ-50 treatment. Following subcutaneous inoculation of nude mice (n = 6 per group) with either PC-3 (panel a) or DU-145 (panel b) human prostate cancer cells, DZ-50 (100mg/kg and 200mg/kg) was administered orally (via oral-gavage) to tumor-bearing-hosts for 14 days (subsequent to palpable tumor formation). Tumor volume was measured daily as described in “Methods”; DZ-50 treatment significantly suppressed prostate tumor volume compared to the vehicle control, P < 0.001. (c) Primary prevention of androgen-independent human prostate cancer by DZ-50. To determine the ability of the new lead drug to prevent prostate cancer development, nude mice were subcutaneously inoculated (n = 6 per group) with PC-3 cells with concurrent exposure (orally) to DZ-50 (200mg/kg) for 2-wks. (d) Prostate cancer xenografts were excised from DZ-50-treated and vehicle control tumor-bearing mice, paraffin-embedded, and tissue sections (6μM) were subjected to immunohistochemical analysis for apoptosis, cell proliferation and tumor vascularity, (panels a and b). The three panels represent TUNEL staining for apoptosis, CD31 immunoreactivity for vascularity, and Ki67 expression for cell proliferation, (Magnification 400x). Quantitative analysis of the results is shown on Table 1.

Figure 5. Suppression of primary tumor growth and prevention of prostate tumor development in human prostate cancer xenograft model by DZ-50

(a, b) Tumor volume of prostate xenografts is reduced following DZ-50 treatment. Following subcutaneous inoculation of nude mice (n = 6 per group) with either PC-3 (panel a) or DU-145 (panel b) human prostate cancer cells, DZ-50 (100mg/kg and 200mg/kg) was administered orally (via oral-gavage) to tumor-bearing-hosts for 14 days (subsequent to palpable tumor formation). Tumor volume was measured daily as described in “Methods”; DZ-50 treatment significantly suppressed prostate tumor volume compared to the vehicle control, P < 0.001. (c) Primary prevention of androgen-independent human prostate cancer by DZ-50. To determine the ability of the new lead drug to prevent prostate cancer development, nude mice were subcutaneously inoculated (n = 6 per group) with PC-3 cells with concurrent exposure (orally) to DZ-50 (200mg/kg) for 2-wks. (d) Prostate cancer xenografts were excised from DZ-50-treated and vehicle control tumor-bearing mice, paraffin-embedded, and tissue sections (6μM) were subjected to immunohistochemical analysis for apoptosis, cell proliferation and tumor vascularity, (panels a and b). The three panels represent TUNEL staining for apoptosis, CD31 immunoreactivity for vascularity, and Ki67 expression for cell proliferation, (Magnification 400x). Quantitative analysis of the results is shown on Table 1.

Figure 5. Suppression of primary tumor growth and prevention of prostate tumor development in human prostate cancer xenograft model by DZ-50

(a, b) Tumor volume of prostate xenografts is reduced following DZ-50 treatment. Following subcutaneous inoculation of nude mice (n = 6 per group) with either PC-3 (panel a) or DU-145 (panel b) human prostate cancer cells, DZ-50 (100mg/kg and 200mg/kg) was administered orally (via oral-gavage) to tumor-bearing-hosts for 14 days (subsequent to palpable tumor formation). Tumor volume was measured daily as described in “Methods”; DZ-50 treatment significantly suppressed prostate tumor volume compared to the vehicle control, P < 0.001. (c) Primary prevention of androgen-independent human prostate cancer by DZ-50. To determine the ability of the new lead drug to prevent prostate cancer development, nude mice were subcutaneously inoculated (n = 6 per group) with PC-3 cells with concurrent exposure (orally) to DZ-50 (200mg/kg) for 2-wks. (d) Prostate cancer xenografts were excised from DZ-50-treated and vehicle control tumor-bearing mice, paraffin-embedded, and tissue sections (6μM) were subjected to immunohistochemical analysis for apoptosis, cell proliferation and tumor vascularity, (panels a and b). The three panels represent TUNEL staining for apoptosis, CD31 immunoreactivity for vascularity, and Ki67 expression for cell proliferation, (Magnification 400x). Quantitative analysis of the results is shown on Table 1.

Figure 6. Inhibition of metastasis of human prostate cancer cells by DZ-50

In the experimental metastasis assay, nude mice (n = 7 per group) were injected with prostate cancer cells PC-3 (2x10⁶) through the tail vein DZ-50 treatment (200mg/kg) was initiated at 10 days post-inoculation for 21 days. Evaluation of the lungs (under dissecting microscope) revealed a significant reduction in the number of metastatic lesions to the lungs in the DZ-50 treated group compared to vehicle control mice; P < 0.05. Arrows indicate metastatic foci on the lungs.