Targeted therapy against EGFR and VEGFR using ZD6474 enhances the therapeutic potential of UV-B phototherapy in breast cancer cells

Abstract

Background: The hypoxic environment of tumor region stimulated the up regulation of growth factors responsible for angiogenesis and tumor proliferation. Thus, targeting the tumor vasculature along with the proliferation by dual tyrosine kinase inhibitor may be the efficient way of treating advanced breast cancers, which can be further enhanced by combining with radiotherapy. However, the effectiveness of radiotherapy may be severely compromised by toxicities and tumor resistance due to radiation-induced adaptive response contributing to recurrence and metastases of breast cancer. The rational of using ZD6474 is to evaluate the feasibility and efficacy of combined VEGFR2 and EGFR targeting with concurrent targeted and localized UV-B phototherapy in vitro breast cancer cells with the anticipation to cure skin lesions infiltrated with breast cancer cells.

Materials and methods: Breast cancer cells were exposed to UV-B and ZD6474 and the cell viability, apoptosis, invasion and motility studies were conducted for the combinatorial effect. Graphs and statistical analyses were performed using Graph Pad Prism 5.0.

Results: ZD6474 and UV-B decreased cell viability in breast cancers in combinatorial manner without affecting the normal human mammary epithelial cells. ZD6474 inhibited cyclin E expression and induced p53 expression when combined with UV-B. It activated stress induced mitochondrial pathway by inducing translocation of bax and cytochrome-c. The combination of ZD6474 with UV-B vs. either agent alone also more potently down-regulated the anti-apoptotic bcl-2 protein, up-regulated pro-apoptotic signaling events involving expression of bax, activation of caspase-3 and caspase-7 proteins, and induced poly (ADP-ribose) polymerase resulting in apoptosis. ZD6474 combined with UV-B inhibited invasion of breast cancer cells in vitro as compared to either single agent, indicating a potential involvement of pro-angiogenic growth factors in regulating the altered expression and reorganization of cytoskeletal proteins in combinatorial treated breast cancer cells. Involvement of combination therapy in reducing the expression of matrix metalloprotease was also observed.

Conclusions: Collectively, our studies indicate that incorporating an anti-EGFR plus VEGFR strategy (ZD6474) with phototherapy (UV-B), an alternative approach to the ongoing conventional radiotherapy for the treatment of infiltrating metastatic breast cancer cells in the skin and for locally recurrence breast cancer than either approach alone.

Figure 1

Effect of radiation (UV-B) and ZD6474 is influenced by VEGF content in breast cancer cells. (A) Cells were grown in serum-free CM for 48 h and VEGF was quantified by using ELISA. ** (p < 0.01), and *** (p < 0.001) indicates the increasing level of significance after performing un-paired t-tests between HMEpC and different breast cancer cells. Dose–response curve of (B) UV-B irradiated and (C) ZD-6474 treated breast cancer cells along with normal mammary HMEpC cells as analyzed by MTT assay after 48 h of post treatment. (D) Dose-dependent growth inhibition of breast cancer cells MCF-7, MDA-MB-468 and HMEpC by UV-B radiation in combination with ZD6474 as analyzed by MTT assay after 48 h of post treatment. Points, mean ± S. E. of three different experiments each performed in quadruplicate.

Figure 2

ZD6474 enhances antiproliferative and apoptotic effects of UV-B radiation. (A) The combinatorial effect of ZD6474 (ZD) and UV-B radiation in decreasing cell viability is attributed to both enhanced antiproliferative and apoptotic effects as shown by decreased cell counts by Trypan blue dye exclusion tests. (B) MCF-7 and (C) MDA-MB-468 cells were treated with ZD6474 (ZD) and/or UV-B radiation, and stained with PI for apoptosis measurements using flow-cytometry. The percentage of apoptotic cells is obtained by gating hypodiploid cells. (D) MDA-MB-468 cells were treated ZD and/or UV-B radiation and stained with DAPI, and observed under CLSM in confocal fluorescence (CF) mode and DIC mode and the merged (CF + DIC) image. Red arrows; apoptotic nuclei, green dashed arrows; membrane blebbing, Arrow heads; intact nuclei. Bars; 10 μM. Representative figures of three independent experiments.

Figure 3

Loss of mitochondrial potential (ΔΨm) in ZD6474 and UV-B irradiated MDA-MB-468. Cells were stained with Rh-123 after treatment with ZD6474 (Z) and/or UV-B radiation (R) along with untreated control (C). Top panel represented (A) dot plot of treated MDA-MB-468. There was an evident change in ΔΨm in both MCF-7 and MDA-MB-468 in combination treatment as shown by the histogram plot (B). Translocation of bax from cytosol to mitochondria vice versa for cytochrome-c translocation was observed in MDA-MB-468 in combination treatment as confirmed by western blotting (C). COX-IV and α-tubulin was taken as loading control for mitochondrial and cytosolic fraction respectively.

Figure 4

ZD6474 modulates UV-B action by altering the expression of caspases and apoptotic proteins. Time vs. pNA formation graphs of (A) MCF-7 and (B) MDA-MB-468 treated ZD6474 (ZD) and/or UV-B radiation for 48 h was studied by monitoring the spectrophotometric readings at 405 nm in order to study the Enzyme kinetics of caspase-3/7 using Ac-DEVD-pNA as substrate. Specific activity of Caspase-3/7 was studied for (C) MCF-7 and (D) MDA-MB-468. Bars (mean ± S.E, n = 3), and * (p < 0.05), ** (p < 0.01) represent levels of significance with respect to control. Western blotting of (E) MCF-7 and (F) MDA-MB-468 cells treated with ZD6474 (Z) and/or UV-B (R) for 48 h and probed with anti-PARP, cyclin E, caspase-3, casapse-7, bcl-2, E-cadherin. β-actin protein expression was used as an internal probe for equal loading. Representative of three independent experiments. Intact PARP, complete arrow; cleaved PARP, dashed arrow; block filled arrow, caspase-3 (MDA-MB-468).

Figure 5

ZD6474 inhibits migration in breast cancer cells in combination with UV-B by inhibiting VEGF signaling pathway. Scratching across a cell monolayer on a 6-well culture plate created a wound and the width of the wound was recorded prior to treatment with ZD6474 and/or UV-B and after indicated h post-treatment. Photomicrograph of ZD6474 and/or UV-B treated (A) MCF-7 and (B) MDA-MB-468 cells after 48 h and 24 h post-treatment respectively. Scale bar, 100 μM. Bars, S.E., three random widths along the wound at indicated h post-treatment of (C) MCF-7 and (D) MDA-MJ B-468 cells. P-values were calculated by using un-paired t-test of the same treatment groups (prior and after stipulated h post-treatment). Breast cancer cells were treated with ZD6474 and/or UV-B and incubated in serum-free CM for 48 h. VEGF level of (E) MCF-7 and (F) MDA-MB-468 was determined by ELISA. All data were derived from a minimum of three independent experiments using different cell preparations. *P < 0.05, comparing levels of untreated control with treated cells.

Figure 6

ZD6474 in combination with UV-B alters cytoskeleton organization and extracellular MMP-9. MCF-7 and MDA-MB-468 cells were treated with ZD6474 (ZD) and/or UV-B for 24 h, and then were immunofluorescently labeled using fluorescently-labeled phalloidin (F-actin-binding protein, green) and DAPI (DNA binding dye, blue). ZD and UV-B effectively blocked cell spreading lamellipodia (white arrow) and induced thick and stress actin fiber (red dashed arrow), whereas combination treatment lead to contraction of cytoplasm and F-actin rings around the periphery of the nucleus as observed under (A) CLSM (white filled arrow head). Bars, 10 μM. ZD blocked lamellipodia and filopodia formations (black arrow), which when combined with UV-B lead to contraction of cytoplasm (black dashed arrow) and formation of membrane blebs and apoptotic bodies (white filled arrow head) in addition with drastic loss of lamellipodia-like structures as evident from the photomicrograph of the surface of breast cancer cell studied under under (B) scanning electron microscope (SEM). Bars, 10 μM. ZD6474 (ZD/Z) in combination with UV-B (R) effectively inhibits MMP-9 activity as evident from (C) Zymograph of CM of MCF-7 and MDA-MB-468 cells treated with ZD and/or UV-B.