Selectively Inducing Cancer Cell Death by Intracellular Enzyme-Instructed Self-Assembly (EISA) of Dipeptide Derivatives

Abstract

Tight ligand-receptor binding, paradoxically, is a major root of drug resistance in cancer chemotherapy. To address this problem, instead of using conventional inhibitors or ligands, this paper focuses on the development of a novel process-enzyme-instructed self-assembly (EISA)-to kill cancer cells selectively. Here it is demonstrated that EISA as an intracellular process to generate nanofibrils of short peptides for selectively inhibiting cancer cell proliferation, including drug resistant ones. As the process that turns the non-self-assembling precursors into the self-assembling peptides upon the catalysis of carboxylesterases (CES), EISA occurs intracellularly to selectively inhibit a range of cancer cells that exhibit relatively high CES activities. More importantly, EISA inhibits drug resistant cancer cells (e.g., triple negative breast cancer cells (HCC1937) and platinum-resistant ovarian cells (SKOV3, A2780cis)). With the IC₅₀ values of 28-80 and 25-44 µg mL^-1 of l- and d-dipeptide precursors against cancer cells, respectively, EISA is innocuous to normal cells. Moreover, using coculture of cancer and normal cells, the selectivity of EISA is validated against cancer cells. Besides revealing that intracellular EISA cause apoptosis or necroptosis to kill the cancer cells, this work illustrates a new approach to amplify the enzymatic difference between cancer and normal cells and to expand the pool of drug candidates for potentially overcoming drug resistance in cancer therapy.

Keywords: anticancer; drug-resistance; enzyme; selectivity; self-assembly.

Figure 1

Illustration of intracellular EISA to inhibit cancer cells and the molecular structure of the precursors and the corresponding self-assembling molecules.

Figure 2

Cell viabilities of multiple cell lines incubated with L-DPT (black curve) or D-DPT (red curve) for 72 h. Cell viabilities of a triple negative breast cancer cell line (HCC1937), a breast cancer cell line (MCF-7), a drug sensitive ovarian cancer cell line (A2780), two drug resistant ovarian cancer cell lines (A2780cis and SKOV3), an adenocarcinoma cell line (HeLa), an osteosarcoma cell line (Saos-2), a drug sensitive sarcoma cell line (MES-SA), a drug resistant sarcoma cell line (MES-SA/Dx5), a melanoma cancer cell line (A375), a hepatocellular carcinoma cell line (HepG2), two glioblastoma cell lines (U87MG and T98G),, a stromal cell line (HS-5) and a neuronal cell line (PC-12) are tested. 10⁴ cells/well were initially seeded in a 96 well plate.

Figure 3

Summary of IC₅₀ values of A) L-DPT and B) D-DPT on multiple cell lines on the third day (* = p≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001 versus IC₅₀ value of precursors on HS-5 cell).

Figure 4

A) Cell viabilities of the stromal cells (HS-5) and ovarian cancer cells (A2780cis and SKOV3) (10⁴ cells/well were initially seeded in a 96 well plate) incubated with the precursor L-DPT (73 μg/mL), D-DPT (37 μg/mL), or CDDP (cisplatin) (37 μg/mL) for 3 days. B) Cell viabilities of the co-cultured SKOV3/HS-5 cells and A2780cis/HS-5 cells incubated with the precursor L-DPT (73 μg/mL) or D-DPT (37 μg/mL) for 3 days. 5000 of each co-cultured cells were initially seeded in a 96 well plate.

Figure 5

Summary of the quantification of esterase activities in multiple cell lines.

Figure 6

Cell viability of SKOV3 cells and A2780cis cells incubated with (A, B) L-DPT alone, or L-DPT + zVAD-fmk (45 μM); (C, D) D-DPT alone, or D-DPT + zVAD-fmk (45 μM) for 72 h (* = p≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001).

Figure 7

Cell viability of SKOV3 cells and A2780cis cells incubated with (A, B) L-DPT alone, or L-DPT + Nec-1 (50 μM); (C, D) D-DPT alone, or D-DPT +Nec-1 (50 μM) for 72 h (* = p≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001).