Anti-proliferative and apoptosis-inducing effects of camptothecin-20(s)-O-(2-pyrazolyl-1)acetic ester in human breast tumor MCF-7 cells

Abstract

Camptothecin-20(s)-O-(2-pyrazolyl-1)acetic ester (CPT6) is a novel semi-synthetic analog of camptothecin. In a previous report, CPT6 possessed higher cytotoxic activity in vitro towards human breast tumor MCF-7 cells than topotecan. In this study, the antitumor activity of CPT6 on the human breast tumor MCF-7 cell line was analyzed using the MTT method. The underlying mechanism of CPT6 action was investigated by analyzing the cell cycle distribution, apoptotic proportion, changes in mitochondrial membrane potential, and intracellular Ca2+ concentration using flow cytometry. Nuclear and mitochondrial morphologies were also observed by laser scanning confocal and transmission electron microscopy. DNA damage was observed in MCF-7 cells treated with CPT6. Low-dose CPT6 had a significant cytotoxic effect and could inhibit proliferation and induce apoptosis in MCF-7 cells, possibly through cell nucleus fragmentation and DNA damage. CPT6 thus appears to display potent antitumor activity against human breast tumor MCF-7 cells via the induction of apoptosis, and may be a useful alternative drug for breast cancer therapy.

Figure 1

Chemical structure of camptothecin-20(s)-O-(2-pyrazolyl-1)acetic ester (CPT6).

Figure 2

The inhibitory effects of three cell lines treated with CPT6 for 72 h.The experiments were performed in triplicate. Data are presented as mean ± SD of three independent experiments.

Figure 3

CPT6 induces apoptosis in MCF-7 cells at 48 h. A: Flow cytometric analysis of MCF-7 cell cycle distribution (excitation wavelength: 351 nm; emission wavelength: 380 nm). (a) Control group; (b) treatment with 0.25 nM CPT6; (c) treatment with 0.5 nM CPT6; (d) treatment with 1 nM CPT6. B: The proportional (%) of Sub-G1 phase population. C: DNA fragmentation of apoptotic MCF-7 cells treated with CPT6 at 0, 0.25, 0.5, 1 and 2 nM for 48 h.

Figure 4

Flow cytometric analysis of CPT6-induced apoptosis in MCF-7 cells for 48h using the annexin V-fluorescein isothiocyanate/propidium iodide method. A: (a) Control group; (b) treatment with 0.25 nM CPT6; (c) treatment with 0.5 nM CPT6; (d) treatment with 1 nM CPT6; (e) treatment with 2 nM CPT6; (f) treatment with 4 nM CPT6. B: the apoptotic cells (%) stained with annexin V positive according to every concentration of CPT6. The data represent the means of three independent experiments and the corresponding standard deviation.

Figure 5

Morphological changes in MCF-7 cells treated with CPT6 for 48 h examined by laser scanning confocal microscopy(excitation: 488 nm, emission: 535 nm). Cultured cells were stained with acridine orange. Bars indicate lunate morphology of chromatin and condensed chromatin. (A) Control group; (B) treatment with 0.5 nM CPT6 (cells were observed at ×60 magnification); (C) control group; (D) treatment with 0.5 nM CPT6 (cells were observed at ×20 magnification ).

Figure 6

Assessment of cell ultrastructures treated with CPT6 for 48 h using transmission electron microscopy. (A) Control group; (B) treatment with 0.5 nM CPT6 (cells were observed at ×5000 magnification); (C) control group; (D) treatment with 0.5 nM CPT6 (cells were observed at ×16,500 magnification).

Figure 7

Flow cytometric analysis of the changes in the mitochondrial membrane potential (ΔΨ m) in MCF-7 cells treated with CPT6 for 48 h. A: (a) Control group; (b) treatment with 0.25 nM CPT6; (c) treatment with 0.5 nM CPT6; (d) treatment with 1 nM CPT6. B: Reduction in ΔΨ m in MCF-7 cells treated with CPT6. The data was obtained according to the percentage of cell number in Q4 quadrant which elicited strong fluorescence, resulting from three independent experiments (* p < 0.05, ** p < 0.01); p value compared with the control group (0 nM CPT6).

Figure 8

Changes in intracellular calcium in MCF-7 cells treated with CPT6 for 48h. A: Black line represents control group; red line represents cells treated with 0.25 nM CPT6; green line represents cells treated with 0.5 nM CPT6; blue line represents cells treated with 1 nM CPT6 (excitation: 488 nm; emission: 525 nm). B: Change in calcium levels in MCF-7 cells. The results were reproducible in three additional independent experiments (* p < 0.05, ** p < 0.01); p value compared with the control group (0 nM).