Effect of Bleomycin on Oral Leukoplakia Cell line- an in Vitro Study

Background Leukoplakia generally refers to a rmly attached white patch on a mucous membrane which is associated with an increased risk of cancer. So it is important to diagnose leukoplakia in early stage. In this study we are trying to treat leukoplakia as a marker for prediagnosis of cancer. Since, bleomycin as a chemotherapeutic agent, is mainly used in the treatment of multiple tumors, and several cancers. Hence in this study effect of bleomycin was studied in D38, a leukoplakia cell line. Methods Percent lethality values of leukoplakia cells after treated with several concentations of bleomycin was studied by acridine orange/ethidium bromide staining (AO/EB), propidium iodide (PI) staining, 3-(4, 5-dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and comet assay. Minimum inhibitory concentration (MIC), lethal concentration 25 (LC 25 ), lethal concentration 50 (LC 50 ), lethal concentration 75 (LC 75 ) and highest permissive concentration (HPC) was calculated from probit computational method. Results From AO/EB staining, PI staining, MTT assay and comet assay the MIC value was 15 mg/L concentration of bleomycin, where as the HPC was found at 100 mg/L of bleomycin. The comet tail length gradually increased from 15 mg/L to 100 mg/L of bleomycin. Percent lethality values were also increased from 15 to 100 mg/L concentration of bleomycin in all the staining protocols as well as in comet assay technique.


Background
Over the years potentially malignant disorders like oral leukoplakia (OL) and oral erythroplakia (OE) are associated with dysplastic cellular changes and hence carry a risk of undergoing malignant transformation leading to oral cancer (OC) [1]. Numerous surgical and nonsurgical modalities have been used for treatment of OL [2]. Some of the non-surgical modalities used for the treatment of OL include photodynamic therapy, beta-carotene, lycopene, or vitamin A [2,3]. Bleomycin a chemotherapeutic agent has also been used for treatment of leukoplakia and other malignant lesions [4,5,6,7,8]. Apart from topical application report intralesional injection of bleomycin into OL lesion has been performed with good results [9]. Another study suggested the delivery of bleomycin into head and neck tumors using iontophoresis [10]. Bleomycin is isolated from the bacteria Streptomyces verticillis [11,12,13], which is a glycopeptide antibiotic. As a chemotherapeutic agent, it is mainly used in the treatment of multiple tumors, testicular carcinomas, lymphomas, and head, neck cancers [14,15]. The biological action of bleomycin is through a sequence-selective, metal-dependent oxidative cleavage of DNA and RNA in the presence of oxygen. It can mediate the oxidative degradation of all major classes of cellular RNAs and inhibition of DNA synthesis [13,16]. Still its mechanism of action has not been elucidated. It has been reported that Bleomycin is responsible for lipid peroxidation and mitochondrial DNA damage [17].
Bleomycin induces G2/M cell cycle arrest, which is important for genomic stability, in cancer cell lines. It causes senescence, apoptosis and mitotic cell death [18,19,20].
Studies have suggested intralesional injection of bleomycin to be highly effective in treatment of warts [21]. Off-label use of intralesional bleomycin is another primary and/or adjunctive therapy for different cutaneous lesions dermatology as several types of cutaneous malignancies, telangiectasias, vascular malformations, hemangiomas, and lesions of leishmaniasis cutisand condyloma acuminate [22]. Studies have also suggested intralesional bleomycin to be more effective in treatment of warts when compared to surgical modalities like cryotherapy [23]. Recent research has revealed that bleomycin is a reliable and safe treatment modality for warts resistant to other therapeutics [24].
Bleomycin along with Adriamycin, vinblastine and dacarbazine is the standard chemotherapy regimen for Hodgkin's, and non-Hodgkin's lymphoma disease squamous cell cancers, sarcoma, melanoma, and testicular cancer. Also it is used to treat malignant pleural effusion and Leukemias [25]. Bleomycin is found to concentrate more in lymphoid tissue and does not cause excessive myelo-suppression, thus is the preferred agent in chemotherapeutic regimens for non hodgkins lymphoma also [26]. Bleomycin is one of the important drugs in induction chemotherapy for testicular cancer [27]. Bleomycin also is a key component in the chemotherapy regimens for cervical and ovarian cancer [28,29].
In this study, effect of bleomycin was studied by using D38 cell line which is a leukoplakia cell line.

Cell Culture
Human leukoplakia cell line D38, were used for this study. The cell line was purchased from NCCS, Pune, India. After getting the cell line, cells were washed thoroughly with PBS and the cell number was counted. The cells were cultured in Dulbecco's modi ed eagle media (DMEM) containing 10% FBS (Fetal Bovine Serum) and 1% penicillin-streptomycin solution in six well culture plate. The cells were kept in an incubator at 5% CO 2 and at 37°C temperature. The cells were maintained by changing the media in two days interval. After getting con uent cells were transferred to 96-well culture plates at concentration of 1X10 4 cells/well for study of effect of bleomycin. The cells were treated with several concentrations of bleomycin (0, 15, 25, 50, 75, 90 and 100 mg/L) and were incubated for 24 hours. After the incubation period, viability of cells were studied by acridine orange/ethidium bromide staining (AO/EB), propidium iodide (PI) staining, 3-(4, 5-dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and comet assay.

AO/EB staining
The AO/EB solution was prepared in PBS at the concentration of 100 µg/mL and is applied to in vitro cultured cells. When observed under the uorescent microscope at 400X, green colour indicated live cells, whereas cells with orange and red colour were recorded as apoptotic and necrotic cells, respectively. AO is taken up by both live and dead cells and emits green uorescence whereas EB is only taken up by dead cells, as the integrity cytoplasmic membrane is lost and it stains nucleus orange. Hence live cells, apoptotic cells and necrotic cells were green, orange and red in appearance, respectively.

PI staining
The working solution for PI staining was prepared after mixing PI stock solution (1 mg/mL) with PBS in the ratio 1:9. For monitoring the viability, an aliquot of 20 µL of cell suspension grown in the presence of bleomycin was mixed properly with an aliquot of 20 µL of the working PI solution. The viable cells would appear green and the nucleus of non-viable cells would appear red in colour, under the uorescent microscope. Toxicity values were obtained after a 24 h of incubation. Probits of observed lethality percentage values were used for analysis of toxicity. The dye, PI binds to DNA by entering dead cells only, which appear red in colour whereas the live cells appear green in uorescent light.

MTT assay
The MTT solution was prepared at the concentration of 5 mg/mL in PBS. After 24 h of bleomycin treatment in a 6-well culture plate, 80 µL of MTT solution was added to each well to study the toxicity effect. The plate was kept in an incubator (37º C, 5% CO 2 ) for 4 h. Then, it was found that the media containing the cells and chemicals converted to blue colour after incubated with MTT. Then gently the mass was centrifuged at 1000 rpm for 10 min at 22º C. The supernatant was removed and the pellet was dissolved in an aliquot of 1 mL 100% dimethyl sulfoxide (DMSO) and kept in the incubator (37º C, 5% Comet assay Single cell gel electrophoresis was carried out to study DNA damage of the treated cell lines. Cultured cells were harvested and used in the alkaline comet assay technique. After coating slides with 1% agarose, the slides were allowed for air dried. Treated cells with different concentrations of bleomycin were centrifuged and pellets were washed with PBS; and the washed cells were mixed with three times the cell volume with the low melting point agarose (LMPA) 1% in sol state. The mixture of cells and LMPA sol was placed over the agarose coated slide that was dried at 4º C for 10 min. The slides were further treated with 1% Triton X 100, 10% DMSO, individually, and were placed in the lysing solution of the mixture of 100 mM Na 2 EDTA, 10 mM Tris, 2.5 mM NaCl (pH, 10), at 4º C for 1 h. The slides were subsequently removed and placed in the electrophoretic buffer consisting of 1 mM Na 2 EDTA and 300 mM NaOH (pH, 13) for 30 min. The slides were subjected to electrophoresis was carried out at 1.

Discussion
Since oral leukoplakia has a poor diagnosis, but it may be fatal due to its development to oral squamouscell carcinoma (OSCC). Risk factors for oral carcinoma have been identi ed, but there are no reliable predictors of the outcome in individual patients with oral leukoplakia. Prevention of leukoplakia to OSCC is important because tramsformation of dysplastic leukoplakiato OSCC is higher in comparison with hyperkeratotic leukoplakia [30]. Chemotherapy may have toxic effects which may be reduced with topical therapy. In a study, it was found that vitamin A has some effect in snuff induced leukoplakia [31,32]. Daily topical application of bleomycin in DMSO for 15-18 days reduces keratinization and dysplasia [8].
In another study, bleomycin at 0.5% and 1.0% has effect in reducing the size and severity of dysplasia after applying once daily for 2 weeks [1,7].
In this study it was found that bleomycin is effective against leukoplakia cell line D38. After treating bleomycin at several concentrations to D38 cell line, it was observed that the MIC values were 15 mg/L of bleomycin, whereas the HPC values were 100 mg/L of bleomycin after 24 hours of incubation, by AO/EB staining, PI staining, MTT assay and comet assay techniques.
Eliminating the use of tobacco and alcohol is the most important aspect of treatment of oral leukoplakia. Now-a-days surgical removal is the common method of management and a choice of treatment by using lasers. But there are some di cult excision sites. In some cases posttreatment complications are also found. So it is di cult to treat the leukoplakia by excision only, in all cases. Hence, bleomycin can be used to prevent the leukoplakia developing to OSCC. Advantages of this approach are easy for application, that doesn't require treatment at a health care centre and relatively low cost as compared to surgical intervention.

Conclusion
Advances in the management of oral leukoplakia will help in prevention of potential progression of oral leukoplakia to SCC through dysplasia. From this study, it is clear that bleomycin can be used as an active drug for the treatment and management of oral leukoplakia.