Table 1.

mtDNA methylation studies in cancer.

Experimental Model	Method	mtDNA Region Investigated	Observation	Reference
Cancer cell lines and tissue specimens from patients with gastric and colorectal cancer	Bisulfite-PCR–single-stranded DNA conformation polymorphism (SSCP)	MT-RNR2, MT-CO1 and MT-CO2 genes	Only unmethylated bands for all analyzed samples were detected	[42]
Human samples of exfoliated cervical lavage positive for HPV16	EpiTYPER and pyrosequencing	Hypervariable segment region	mtDNA methylation was generally low, although few sites showed differences in CpG methylation by disease state	[69]
Colorectal cancer tissues and paired adjacent non-cancerous tissues	Methylation-specific PCR	D-loop region	Hypomethylation of D-loop and increased MT-ND2 protein expression in cancer tissues	[58]
Colorectal cancer tissues and paired adjacent non-cancerous tissues	Methylation-specific PCR	D-loop region	Hypomethylation of D-loop and increased MT-ND2 protein and mtDNA copy number expression in cancer tissues	[55]
Five colorectal cancer cell lines treated with the DNA demethylating agent 5-azacytidine	Sequenom MassARRAY	D-loop region	5-azacytidine induced D-loop demethylation and increased mtDNA copy number content	[70]
Peripheral blood from fifteen female individuals of five families with one breast cancer patient	Bisulfite sequencing	Whole-mtDNA genome methylation	Eight aberrant D-loop methylation sites were correlated with breast cancer. Evidence that mtDNA methylation pattern was maternally inherited.	[71]
Adenoma and normal mucosa paired samples	Whole-genome bisulfite sequencing (WGBS)	Whole-mtDNA genome methylation	Methylation in mtDNA was low in both normal and adenoma tissue and was not associated with mitochondrial gene transcription	[72]
Cellular models of glioblastoma and osteosarcoma	Whole-mtDNA bisulfite sequencing, pyrosequencing and 5-mC and 5-hmC immunoprecipitation	Whole-mtDNA genome methylation	The mtDNA methylation levels decreased during tumor progression along with increased mtDNA copy number. D-loop methylation negatively correlated with MT-ND5 and MT-ND6 transcription during the tumorigenesis of osteosarcoma cells.	[73]
Normal breast epithelial cells (MCF10A), human breast cancer cells (MCF7), primary human liver (hepatocytes), human hepatocarcinoma cells (HepG2) and primary derived human breast cancer cells (HMEC)	Bisulfite sequencing	Whole-mtDNA genome methylation	Globally, the L-strand displayed a higher degree and frequency of methylation compared to the H-strand. The highest frequency of methylation was detected within a CpT or CpC dinucleotide context, whereas methylation of CpG sites was the least frequently methylated dinucleotide. Methylation patterns display notable differences between normal and cancer cells.	[61]
Cisplatin sensitivity in oral squamous cell carcinoma (OSCC) cell lines, namely, SAS and H103, and stem cell-like tumor spheres derived from SAS	Nanopore sequencing (MinION)	Whole-mtDNA genome methylation	SAS tumor spheres and H103 cells were less sensitive to cisplatin than SAS. Cells with lower mtDNA content were less responsive to cisplatin. Enhanced cisplatin resistance in stem cell-like tumor spheres was not influenced by methylation status of the mitochondrial genome. Hypermethylated of MT-CO1 and MT-CYB in H103 with concomitant higher expression levels of most of the mitochondrial genes, including MT-CO1 and MT-CYB.	[74]
Matched tumor/normal samples of liver (n = 10 pairs) and head and neck (n = 6 pairs) cancers	Nanopore sequencing (MinION and PromethION systems)	Whole-mtDNA genome methylation	Differentially methylated sites between tumor and their matched adjacent tissues detected in head and neck, but not in liver	[75]