Indolo-pyrido-isoquinolin based alkaloid inhibits growth, invasion and migration of breast cancer cells via activation of p53-miR34a axis

Abstract

The tumor suppressor p53 plays a critical role in suppressing cancer growth and progression and is an attractive target for the development of new targeted therapies. We synthesized several indolo-pyrido-isoquinolin based alkaloids to activate p53 function and examined their therapeutic efficacy using NCI-60 screening. Here, we provide molecular evidence that one of these compounds, 11-methoxy-2,3,4,13-tetrahydro-1H-indolo[2',3':3,4]pyrido[1,2-b]isoquinolin-6-ylium-bromide (termed P18 or NSC-768219) inhibits growth and clonogenic potential of cancer cells. P18 treatment results in downregulation of mesenchymal markers and concurrent upregulation of epithelial markers as well as inhibition of migration and invasion. Experimental epithelial-mesenchymal-transition (EMT) induced by exposure to TGFβ/TNFα is also completely reversed by P18. Importantly, P18 also inhibits mammosphere-formation along with a reduction in the expression of stemness factors, Oct4, Nanog and Sox2. We show that P18 induces expression, phosphorylation and accumulation of p53 in cancer cells. P18-mediated induction of p53 leads to increased nuclear localization and elevated expression of p53 target genes. Using isogenic cancer cells differing only in p53 status, we show that p53 plays an important role in P18-mediated alteration of mesenchymal and epithelial genes, inhibition of migration and invasion of cancer cells. Furthermore, P18 increases miR-34a expression in p53-dependent manner and miR-34a is integral for P18-mediated inhibition of growth, invasion and mammosphere-formation. miR-34a mimics potentiate P18 efficacy while miR-34a antagomirs antagonize P18. Collectively, these data provide evidence that P18 may represent a promising therapeutic strategy for the inhibition of growth and progression of breast cancer and p53-miR-34a axis is important for P18 function.

Keywords: Breast cancer; EMT; Indolo-pyrido-isoquinolin based alkaloid; Invasion; miR34a; p53.

Figure 1

Evaluation of sempervirine analogs including P18 in NCI‐60 screening. (A) Sempervirine analogs were synthesized. IUPAC nomenclature of analogs studied. (B) Analogs were evaluated in the NCI‐60 screen. Heat map shows the log10 GI50 values for each sempervirine analog in the NCI‐60 screen, where high intensity (red) cells indicate high activity and low intensity (blue) cells indicate low activity.

Figure 2

P18 inhibits breast cancer growth, modulates epithelial and mesenchymal markers and inhibits migration and invasion of breast cancer cells. (A) MCF7, HBL100, MCF10A and HMEC cells were treated with various concentration of P18 for 24, 48 and 72 h as indicated and subjected to XTT cell proliferation assay. *p < 0.05 compared with untreated controls. Vehicle‐treated cells are denoted with C. (B) Clonogenicity of breast cancer cells treated with various concentrations of P18 (as indicated). Representative images are shown. (C) TUNEL staining of breast cancer cells treated with P18. Bar graph shows number of TUNEL positive cells. *p < 0.001 compared with untreated controls (C). (D) Invasion potential of MCF7 cells treated with 5 μM P18 was examined using Matrigel‐invasion assay. (E) Breast cancer cells were treated with 5 and 10 μM P18 and subjected to spheroid migration assay. (F) Breast cancer cells were treated with 5 μM P18 followed by scratch‐migration assay. (G) MCF‐7 cells were treated with 5 μM P18 for indicated time intervals. Total RNA was isolated and expression level of epithelial (E‐cadherin and CK‐18) marker genes, mesenchymal (Fibronectin and Vimentin) marker genes and EMT‐related transcription factors (Snail and Zeb1) was analyzed. Actin was used as control.

Figure 3

P18 inhibits TGFβ/TNFα‐induced epithelial‐mesenchymal transition in mammary epithelial cells. (A) MCF10A cells were treated with vehicle control (control), TGFβ + TNFα (10 ng/mL of each, TT), 5 μM P18 or TGFβ + TNFα+P18 for 72 h. Morphological changes associated with EMT are shown in phase‐contrast images. The presence of spindle‐shaped cells, increased intercellular separation and pseudopodia were noted in TGFβ + TNFα‐treated cells but not in P18 treated or TGFβ + TNFα + P18‐treated cells. (B) MCF10A cells were treated as in A, total RNA was isolated and expression of mesenchymal markers (fibronectin, vimentin) and EMT‐related transcription factors (SNAIL, ZEB2) was analyzed. Actin was included as control. (C) MCF10A cells were treated as in A, and subjected to immunofluorescence analysis of Snail, Slug and Zeb1 (Magnification 1000×).

Figure 4

P18 suppresses mammosphere‐formation potential and acquisition of stem‐like properties in breast cancer cells. (A) MCF7 and HBL‐100 cells were treated with 5 and 10 μM P18 and subjected to mammosphere formation. Vehicle treated cells are denoted as (C). The graph shows the number of mammospheres. (B) MCF7 cells were treated with 5 μM P18 for indicated time intervals, total RNA was isolated and expression level of stemness marker (Oct4, Nanog and Sox2) genes was analyzed. Actin was used as control. (C) MCF7 and HBL100 cells were treated with 5 μM P18 for indicated time intervals, total protein was isolated and expression level of stemness marker (Oct4, Nanog and Sox2) genes was analyzed using immunoblot analysis. Actin was used as control. (D) MCF7 cells were treated with vehicle control (control), TGFβ + TNFα (10 ng/mL of each, TT), 5 μM P18 or TGFβ + TNFα+P18 and subjected to immunofluorescence analysis of Oct4 (Magnification 1000×).

Figure 5

P18 induces p53 expression, phosphorylation and increases nuclear localization of p53 in breast cancer cells. p53 plays an important role in mediating biological effects of P18. (A) MCF7 cells were treated with 5 μM P18 for indicated time‐intervals, total RNA was isolated followed by RT‐PCR analysis for p53, p27 and p21. Actin was used as control. (B) MCF7 cells were treated with 5 μM P18 for indicated time‐intervals, total protein was isolated followed by immunoblot analysis for p53, p27 and p21. Actin was used as control. (C) MCF7 cells were treated with 5 μM of P18 followed by immunofluorescence microscopy against phosphorylated p53 at Serine 15 (p‐p53‐S15) (stained with Cy5, red). Nuclei were stained with DAPI (blue). (Magnification 1000×). (D) MCF7 cells were treated with 5 μM P18 for indicated time‐intervals, total protein was isolated followed by immunoblot analysis for p53‐Thr18 and MDM2. Actin was used as control. (E) Total protein was isolated from HCT116‐p53+/+ and HCT116‐p53−/− cells and immunoblotted for p53 expression. (F)Total RNA was isolated from HCT116‐p53+/+ and HCT116‐p53−/− cells treated with 5 and 10 μM of P18 as indicated and expression of mesenchymal markers (fibronectin, vimentin), epithelial marker (E‐cadherin) and EMT‐related transcription factors (Snail, Slug and Zeb1) was analyzed. Actin was included as control. (G) HCT116‐p53+/+ and HCT116‐p53−/− cells treated with 5 and 10 μM of P18 as indicated and subjected to scratch‐migration assay. Bar graphs show fold change in migration. *p < 0.01, compared with untreated controls. (H) HCT116‐p53+/+ and HCT116‐p53−/− cells treated with 5 μM of P18 as indicated and subjected to invasion assay. Bar‐graph shows numbers of cells invaded through Matrigel. Representative pictures are shown. *p < 0.05, compared with untreated controls. (I) MCF7 cells were transfected with control‐si or p53‐si as indicated, treated with 5 and 10 μM of P18 and subjected to invasion assay. *p < 0.005, compared with untreated controls. (J) MCF7 cells were transfected with control‐si or p53‐si as indicated, treated with 5 and 10 μM of P18 and subjected to scratch‐migration assay. *p < 0.005, compared with untreated controls.

Figure 6

p53 plays an integral role in P18‐mediated upregulation of miR‐34a expression. miR‐34a is integral for P18‐mediated inhibition of growth, invasion and mammosphere formation. (A) TaqMan RT‐PCR analysis of miR‐34a expression in MCF‐7 and HBL‐100 cells treated with 5 and 10 μM P18 as indicated. C denotes vehicle‐control. *p < 0.05, compared with untreated controls. (B) MCF7 cells were transfected with control‐si or p53‐si as indicated, treated with 5 and 10 μM of P18 and total RNA was isolated followed by TaqMan RT‐PCR analysis of miR‐34a expression. C denotes vehicle‐control. *p < 0.05, compared with untreated controls. (C) MCF7 and HBL100 cells were transfected with miR‐34a mimic or antagomir (inhibitor), treated with P18 as indicated followed by XTT cell proliferation assay. *p < 0.001 compared with untreated controls. **p < 0.005 compared with P18‐treated cells. Vehicle‐treated cells are denoted with C. (D) MCF7 and HBL100 cells were transfected with miR‐34a mimic or antagomir (inhibitor), treated with P18 as indicated followed by Matrigel‐invasion assay. *p < 0.01 compared with untreated controls. **p < 0.001 compared with P18‐treated cells. Vehicle‐treated cells are denoted with C. (E) MCF7 and HBL100 cells were transfected with miR‐34a mimic or antagomir (inhibitor), treated with P18 as indicated followed by mammosphere assay. Data shows numbers of mammosphere. *p < 0.05, compared with untreated controls. **p < 0.01 compared with P18‐treated cells.