J Vet Sci.  2016 Jun;17(2):235-242. 10.4142/jvs.2016.17.2.235.

Hematoporphyrin monomethyl ether combined with He-Ne laser irradiation-induced apoptosis in canine breast cancer cells through the mitochondrial pathway

• Li H
• Tong J
• Bao J
• Tang D
• Tian W
• Liu Y

• KMID: 2413173
• DOI: http://doi.org/10.4142/jvs.2016.17.2.235

Abstract

Hematoporphyrin monomethyl ether (HMME) combined with He-Ne laser irradiation is a novel and promising photodynamic therapy (PDT)-induced apoptosis that can be applied in vitro on canine breast cancer cells. However, the exact pathway responsible for HMME-PDT in canine breast cancer cells remains unknown. CHMm cells morphology and apoptosis were analyzed using optical microscope, terminal deoxynucleotidyl transferase dUTP nick end labeling fluorescein staining and DNA ladder assays. Apoptotic pathway was further confirmed by Real-time-polymerase chain reaction and Western blotting assays. Our results showed that HMME-PDT induced significant changes in cell morphology, such as formation of cytoplasmic vacuoles and the gradual rounding of cells coupled with decreased size and detachment. DNA fragmentation and cell death was shown to occur in a time-dependent manner. Furthermore, HMME-PDT increased the activities of caspase-9 and caspase-3, and released cytochrome c from mitochondria into the cytoplasm. HMME-PDT also significantly increased both mRNA and protein levels of Bax and decreased P53 gene expression in a time-dependent manner, while the mRNA and protein expression of Bcl-2 were repressed. These alterations suggest that HMME-PDT induced CHMm cell apoptosis via the mitochondrial apoptosis pathway and had anti-canine breast cancer effects in vitro.

Keyword

CHMm cells; apoptosis; hematoporphyrin monomethyl ether; mitochondrial pathway; promising photodynamic therapy

MeSH Terms

Animals
*Apoptosis/radiation effects
Breast Neoplasms/etiology/surgery/*veterinary
Caspase 3/metabolism
Caspase 9/metabolism
Cell Line, Tumor
Cytochromes c/metabolism
Dog Diseases/etiology/*surgery
Dogs
Gene Expression/drug effects/radiation effects
Helium
Hematoporphyrins/*pharmacology
*Lasers
Mitochondria/*drug effects/metabolism/*radiation effects
Neon
Photosensitizing Agents/pharmacology
Hematoporphyrins
Photosensitizing Agents
Helium
Neon
Cytochromes c
Caspase 3
Caspase 9

Figure

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  Fig. 1 Changes in cell morphology determined following hematoporphyrin monomethyl ether (HMME)-promising photodynamic therapy (PDT) treatment. CHMm cells were treated as indicated, after which images were acquired using a light microscope. Arrows indicate cells contained cytoplasmic vacuoles. Arrowheads show cells became round and smaller and detached. Scale bars = 100 µm.

• 

  Fig. 2 TUNEL fluorescein staining to detect DNA fragmentation in CHMm cells following PDT–HMME treatment. Green fluorescence indicates DNA fragmentation. HMME-PDT treatments resulted in increased numbers of TUNEL-positive cells at all tested time points (6, 12, 24 and 48 h). Scale bars = 100 µm.

• 

  Fig. 3 DNA ladder assays detected DNA fragmentation in CHMm cells following PDT–HMME treatment. The ladder indicates DNA fragmentation. The ladder appeared first at 3 h after exposure, then became increasingly prominent with lengthening of exposure time.

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  Fig. 4 HMME-PDT induced changes in caspase 3, 9 and Cyto C in canine breast cancer cells. (A) HMME-PDT induced the release of Cyto C at 6, 12, 24, and 48 h after PDT. (B and C) The activities of caspase-9 and caspase-3 were remarkably stimulated from 6 h and 12 h, respectively, when compared with the mock treated cells (n = 3, mean ± SD).

• 

  Fig. 5 Relative gene and protein expression in the mitochondrial apoptotic pathway. (A) The treatment up-regulated the mRNA of P53 and Bax and down-regulated Bcl-2. (B and C) Bcl-2 expression was down-regulated and Bax expression was up-regulated, further decreasing the ratio of Bcl-2 to Bax.

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