Genetic analysis of a novel antioxidant multi-target iron chelator, M30 protecting against chemotherapy-induced alopecia in mice

Abstract

Background: Chemotherapy-induced alopecia has been well documented as a cause of distress to patients undergoing cancer treatment. Almost all traditional chemotherapeutic agents cause severe alopecia. Despite advances in the treatment of chemotherapy-induced alopecia, there is no effective treatment for preventing chemotherapy-induced alopecia.

Methods: In the present study, we investigated the potential role of a multi-target iron chelator, M30 in protecting against cyclophosphamide-induced alopecia in C57BL/6 mice implanted with an osmotic pump. M30 enhanced hair growth and prevented cyclophosphamide-induced abnormal hair in the mice. Furthermore, we examined the gene expression profiles derived from skin biopsy specimens of normal mice, cyclophosphamide-treated mice, and cyclophosphamide treated mice with M30 supplement.

Results: The top genes namely Tnfrsf19, Ercc2, Lama5, Ctsl, and Per1 were identified by microarray analysis. These genes were found to be involved in the biological processes of hair cycle, hair cycle phase, hair cycle process, hair follicle development, hair follicle maturation, hair follicle morphogenesis, regulation of hair cycle.

Conclusion: Our study demonstrates that M30 treatment is a promising therapy for cyclophosphamide-induced alopecia and suggests that the top five genes have unique preventive effects in cyclophosphamide-induced transformation.

Keywords: Alopecia; Anti-oxidant; Chemotherapy; Cyclophosphamide; M30; Microarray.

Fig. 1

Macroscopic and histological effects of M30 on CTX-induced alopecia in C57BL/6 mice. The mice were depilated using wax strips and intraperitoneally injected with CTX with or without M30 supplement (see Methods section). After CTX treatment, shaved skin of the normal, CTX-treated, and M30-supplemented CTX-treated mice was photographically observed 2 weeks after CTX treatment. a Representative images of dorsal skin of normal mice (Normal), CTX-treated mice (CTX), and M30-supplemented CTX-treated mice (M30 + CTX) are shown. The sample tissue was sliced into 5-μm-thick transverse sections and these sections (n = 5) were observed using a confocal microscope. b Representative images of skin of normal mice (Normal), CTX-treated mice (CTX), and M30-supplemented CTX-treated mice (M30 + CTX) are shown. Scale bar = 100 μm

Fig. 2

Representative MA plot of changes and hierarchical clustering analyses in gene expression levels after CTX and M30 treatment. a Hierarchical cluster analysis of all samples in the gene expression microarray. Genes that were upregulated relative to control are shown in red and those that were downregulated are shown in green. The expression levels of these genes were altered ≥1.5-fold or ≤ 0.666-fold in the CTX/Normal, MC/CTX, and MC/Normal condition (p < 0.05). b MA plots comparing each of the three data sets for a representative sample. Gene expression profiles from the normal mouse skin compared with CTX-treated mouse skin (CTX/Normal), normal mouse skin compared with M30-supplemented CTX-treated mouse skin (MC/CTX), and CTX-treated mouse skin compared with M30-supplemented CTX-treated mouse skin (MC/Normal). MA plot where M = log (Cy5/Cy3) is the log ratio of the two dyes used in the hybridization, and A = [log (Cy5) + log (Cy3)]/2 is the average of the log intensities. c Venn diagram showing the number of genes regulated by CTX, or CTX with M30. The number of upregulated, contra-regulated, and downregulated genes that responded commonly or uniquely to the treatments is shown in red arrows, red texture, and blue arrows, respectively

Fig. 3

The main functional categories showing significantly changed hair-related genes after CTX and M30 treatment. a-d The number of differentially expressed genes that were demonstrated in the following GO terms is indicated: hair cycle, hair cycle phase, hair cycle process, hair follicle development, hair follicle maturation, hair follicle morphogenesis, and regulation of hair cycle. Upregulated or downregulated genes in the following conditions of a CTX/Normal, b MC/CTX, and c MC/Normal are shown. d M30-recovered genes against CTX treatment in conditions 1 and 2 (see Table 2) are shown

Fig. 4

Interaction networks of five significantly changed hair-related genes after CTX and M30 treatment. Five target gene interaction networks were constructed by Cytoscape software (version 2.7.0; http://www.cytoscape.org). The top five target genes were screened according to the rank of target gene pair-specific context score. The genes in red nodes indicate CTX upregulated genes, blue nodes indicate downregulated genes, and the genes in edges indicate the interactive 25 genes

Fig. 5

The main functional categories showing significantly changed genes after CTX and M30 treatment. a-c The number of differentially expressed genes that were demonstrated in the following GO terms is indicated: angiogenesis-, aging-, cell proliferation-, cell migration-, cell death-, apoptosis-, inflammation-, RNA splicing-, extracellular matrix-, immune response-, and secretion-related genes. a CTX/Normal, b MC/CTX, and c MC/Normal

Fig. 6

Interactions networks of five significantly changed genes after CTX and M30 treatment. Target gene interaction networks were constructed by Cytoscape software (version 2.7.0; http://www.cytoscape.org). The genes in red nodes indicate CTX upregulated genes, blue nodes indicate downregulated genes, and the genes in edges indicate the interactive 25 genes