A Priori Activation of Apoptosis Pathways of Tumor (AAAPT) technology: Development of targeted apoptosis initiators for cancer treatment

Abstract

Cancer cells develop tactics to circumvent the interventions by desensitizing themselves to interventions. Amongst many, the principle routes of desensitization include a) activation of survival pathways (e.g. NF-kB, PARP) and b) downregulation of cell death pathways (e.g. CD95/CD95L). As a result, it requires high therapeutic dose to achieve tumor regression which, in turn damages normal cells through the collateral effects. Methods are needed to sensitize the low and non-responsive resistant tumor cells including cancer stem cells (CSCs) in order to evoke a better response from the current treatments. Current treatments including chemotherapy can induce cell death only in bulk cancer cells sparing CSCs and cancer resistant cells (CRCs) which are shown to be responsible for high recurrence of disease and low patient survival. Here, we report several novel tumor targeted sensitizers derived from the natural Vitamin E analogue (AMP-001-003). The drug design is based on a novel concept "A priori activation of apoptosis pathways of tumor technology (AAAPT) which is designed to activate specific cell death pathways and inhibit survival pathways simultaneously and selectively in cancer cells sparing normal cells. Our results indicate that AMP-001-003 sensitize various types of cancer cells including MDA-MB-231 (triple negative breast cancer), PC3 (prostate cancer) and A543 (lung cancer) cells resulting in reducing the IC-50 of doxorubicin in vitro when used as a combination. At higher doses, AMP-001 acts as an anti-tumor agent on its own. The synergy between AMP-001 and doxorubicin could pave a new pathway to use AAAPT leading molecules as neoadjuvant to chemotherapy to achieve better efficacy and reduced off-target toxicity compared to the current treatments.

Scheme 1. Structures of AMP-001, AMP-002 and AMP-003.

Fig 1

A) Representative IC-50 synergy curve (% of untreated control cell death Vs Doxorubicin dose) for AMP-001, AMP-002 and AMP-003, N = 4, P < 0.05, B) Representative combination index (CI) calculated at fractional kill Fa-50, Fa-75 and Fa-90 doses generating Isobologram (< 1.00, synergistic), CI range for combination of AMP-001 with doxorubicin or paclitaxel (see Table 1), N = 4, p< 0.05, C: Photomicrographs of synergistic effect of AMP-001 with doxorubicin in TNBC MDA-MB-231 cancer cells.

Fig 2

Kinetics of reactive oxygen species (ROS), A) Triple negative breast cancer (TNBC) cells (MDA-MB-231) and B) prostate cancer cells (PC3) were incubated with α-TOS (60 μM) and AMP compounds (30 μM) for 6 hrs, and ROS formation was evaluated by DCFDA (20 μM) fluorescent dye, and analyzed by a fluorescence plate reader (Infinite F200 PRO, Sunrise, Tecan, Männedorf, Swiss), N = 4, p < 0.03.

Fig 3. Lysosomal instability evidenced by release of acridine orange in AMP-001/002 treated (30 mM) TNBC MDA-MB-231 and in PC3 cells.

TNBC MDA-MB-231 cells (A, B, C) and PC3 prostate cells (D, E, F) were treated with AMP-001/002 respectively and uptake of acridine orange dye was assessed by confocal microscopy, N = 4, p <0.006.

Fig 4. “Treatment with AMP-001/002 causes release of mitochondrial AIF-1 (Mito) into the cytoplasm (Cyto)”.

TNBC MDA-MB-231 cells were treated with AMP-001/002 (30 μM), or positive control α-TOS (60 μM) and protein levels of AIF-1 were assessed in mitochondrial and cytoplasmic cellular fractions with Actin as a reference control (A). AIF-1 levels are quantified in bar graph (B), N = 5, p < 0.006.

Fig 5

A) Quantitative Real Time RT-PCR Analysis of Ratio Bax and Bcl2 in MDA-MB-231 Cells Post Treated with AMP-001 normalized to B-Actin and B) Normalized to Actin, C) Western Blotting of Bax and Bcl2 protein released post treatment of AMP-001, Doxorubicin and combination.

Fig 6. Treatment of CSC enriched cells with AMP-001 negatively affects cancer stem cell activity and reduces mammosphers (precursor for self-renewal of CSCs) compared to control in vitro.

Mammospheres were measured by microscopy following treatment with AMP-001 in two different CSC enriched cell lines, MCF-10A-ER-SRc and TMD-231, n = 4 experiments, p< 0. 04, SD = 7.8, A & C: Untreated Controls (3 Views, Same Scale).

Fig 7

A) Translocation of p65 in MDA-MB-231 cancer cells from nucleus to cytoplasm by Western Blotting (See arrow) post treatment by AMP-001/002/003, B) Deliberate treatment of MDA-MB-231 cancer cells with Alfa-TNF, NF-kB activator followed by translocation of p65 from nucleus to cytoplasm for control compared to AMP-00/003 treatment in presence of Alfa-TNF (and C) quantitative data on p65 translocation from nucleus to cytoplasm-hallmark of NF-kB inhibition, D) p65 protein translocation in presence of Alfa-TNF for AMP-001, AMP-002 and AMP-003.

Fig 8

A: Increased expression of CD95 (40 KDa) band in Western Blotting upon treatment of MDA-MB-231 cancer cells by AMP-001 and AMP-002, B) total and cleaved PARP levels by dose dependent treatment of MDA-MB-231 cells by AMP-001. N = 4 P<0.001 for A, p < 0.004 for B, Beta-Actin and GAPDH are standard controls.

Fig 9

Photomicrographs of induced pluripotent stem cells (iPSc) derived cardiomyocytes post treated with A) DMSO control, B) 250 μM of AMP-001, C) 10 μM of Doxorubicin, D) 100 μM of AMP-001, E) IC-50 graph normalized to 1 corresponding to 100% viability, Insert Table IC-50 for Vitamin E, Sorafinib added for comparison.

Fig 10. AMP-001 monotherapy treatment in TNBC mouse model results in reduced tumor growth and size.

Mice (n = 8/group were injected i.p with AMP-001 and the tumor regression was monitored through Bioluminescent Imaging signal (A) and tumor volume measurement, P <0.05, (B). Schedule: Three times a week for two weeks. Change in tumor volume for treated tumor as a percentage of tumor growth (C) H&E staining was performed on non-target organ heart specimens to demonstrate intactness of cellularity (C-3 & C4) or destruction of tissue structure in tumor respectively (C2) compared to untreated control (C1), C: 2–4 Magnification X 400, n = 5, p < 0.001.