Role of Mycoplasma in the Initiation and Progression of Oral Cancer.

Mycoplasma represents a unique group of bacteria measuring about 0.2-0.8 µm in diameter. Due to the absence of a cell wall, Mycoplasma varies in size and shape. A number of these Mycoplasma species such as Mycoplasma salivarium, Mycoplasma orale, Mycoplasma faucium, Mycoplasma buccale, Mycoplasma fermentans, Mycoplasma lipophilum, and Mycoplasma pneumonia form a part of the normal microbial flora of the oropharynx.1-3 Among these M. salivarium and M. orale are isolated at a higher rate in the mouth including the saliva, gingival sulci, dental plaque, and periodontal pockets. Due to its small genome, Mycoplasma lacks the necessary metabolic option for self-survival and replication.1-3 Thus, it relies on a host cell for its metabolic needs (parasitic organism).

The location of Mycoplasma was assumed to be on the surface of cells. However, recent data have confirmed its intracellular location using both in-vitro and in-vivo techniques. Now the question arises as to the long-term effects of a parasitic organism like Mycoplasma on the host cell. The mere presence of Mycoplasma in oral cancer tissues does not merit a designation as a carcinogen, as it forms a part of the normal microbial flora. There have been various studies reporting isolation of a greater percentage of Mycoplasma species in various infectious, neoplastic tissues, and body fluids than in a healthy individual. Few researchers have even correlated a possible association of Mycoplasma with AIDS.4

To answer these queries in-vitro studies were conducted to observe the effects of Mycoplasma on cell lines. Tsai et al.5 in 1995 infected cultured mouse embryo cells, C3H/10T1/2 (C3H) with M. fermentans and Mycoplasma penetrans. They found that Mycoplasma species did not have any detectable effect on the cell morphology or the growth pattern of the cell lines up to five passages (5 weeks). M. fermentans and M. penetrans had a reversible effect on the cultured cells during the 6th and 15th passages respectively. The changes consisted of the following: The cells lost cell to cell contact, assumed spindle morphology and exhibited growth in multiple layers. The reversible nature of these changes was elicited by treating the cells with three cycles (1 week per cycle) of ciprofloxacin. Eradication of Mycoplasma was confirmed by polymerase chain reaction (PCR). Most of the transformed cells reverted back to their original morphology and growth pattern (flat monolayer growth). The cells subjected to more than 18 passages failed to revert back to its previous morphology/growth pattern, indicating an irreversible change. Thus, persistent infection with Mycoplasma induced a multistep step carcinogenesis.5

Barykova et al.6 in 2011 investigated the role of Mycoplasma hominis in prostate cancer. They found that M. hominis was detected 3 times higher in patients with prostate cancer in comparison to patients with benign prostatic hyperplasia. Further the prostate specific antigen levels were significantly elevated in subjects positive for M. hominis. Correlating these data, they suggested a causal link of Mycoplasma in the development of prostate cancer.6 Logunov et al.7 analyzed the mechanism of Mycoplasma induced carcinogenesis. They compared the effects of Mycoplasma infection on apoptotic regulators like p53 and nuclear factor (NF)-κB. They infected mouse Balb 3T3 cells with Mycoplasma arginini. They noticed a dramatic decrease in activation of p53 with a constitutive activation of NF-κB. This pattern of altered expression was consistent with many human cancers.7 Thus, Mycoplasma infected cells were able to evade apoptosis by inhibiting p53 which in turn rendered the cells susceptible to H-Ras. The control cells not infected with Mycoplasma underwent a p53 controlled cell arrest.7 Feng et al.8 observed that the 32D murine myeloid cell line undergoes apoptosis following withdrawal of interleukin-3 (IL-3). However, when infected with Mycoplasma species the 32D cell line evaded apoptosis even in the absence of IL-3.

Though sufficient studies have demonstrated the malignant potential of Mycoplasma in prostate cancer, myeloid cell lines, and mouse embryo cells, there was no substantial evidence linking Mycoplasma to oral cancer.8 Mizuki was the first to formulate a correlation between oral leukoplakia and M. salivarium.9 They observed a small granular bisbenzimide stained fluorescent structures in the cytoplasm of oral leukoplakia cells. Based on its morphology and size they presumed it to represent a Mycoplasma species. Following the observation of Mizuki et al. several light and electron microscopic studies were conducted to reveal the presence of Mycoplasma in the oral leukoplakia cells. None of the studies revealed their presence spiraling debate as to Mizuki et al. findings. To confirm their earlier results Mizuki et al. performed an immunohistochemical analysis to detect Mycoplasma in oral leukoplakia cell.10 They utilized both a polyclonal and a monoclonal antibody specific for M. salivarium. The result showed a substantial increase in the presence of Mycoplasma within the cytoplasm of oral leukoplakia in comparison to control (normal oral mucosa). Immunoelectron microscopy demonstrated electron dense particles in the cytoplasm of oral leukoplakia cells confirming their intracellular location. The presence of M. salivarium was reconfirmed by PCR. PCR produced a fragment of about 150 bp. The NCBI/BLAST databases indicated a 100% match to M. salivarium.10

Based on the consistent demonstration of Mycoplasma in oral leukoplakia, Mizuki et al. proposed a causal role for M. salivarium in the initiation of oral leukoplakia. The proposal was based on the following data: (a) Salivary IL-6 and tumor necrosis factor alpha are reported to be expressed at a higher level in oral leukoplakia,11 (b) several studies including that of Drexler and Uphoff have found that Mycoplasma induced increased cytokine expression (as noticed in oral leukoplakia) in addition to various chromosomal alterations,12 (c) further the intensity of the polyclonal and monoclonal antibody of M. salivarium was higher in areas with thicker cornified layer (hyperkeratosis, representing the initial stage of oral leukoplakia). At present, there is a lack of in-vivo studies illustrating the pathogenesis of Mycoplasma in oral potentially malignant disorders. In vivo studies using animal models like the Syrian hamster cheek pouch may aid us in understanding the molecular pathway of Mycoplasma induced oral carcinogenesis, which in turn will provide us with vital diagnostic markers and potential therapeutic targets.