Poster 031: Evaluation of the Type of Cell Death Induced by Mitomycin C in Human Oral Squamous Cell Carcinoma Cell Lines

Abstract

Statement of the ProblemPeplomycin (PEP) and Mitomycin C (MMC) are used very often as neo-adjuvant chemotherapy before radical surgery. In contrast to hundreds of reports about the effect of MMC in vitro, there is little comparative study of cytotoxicity against bothhuman oral squamous cell carcinoma (SCC) cell lines and human oral normal cell lines. In the past, many of such studies have dealt with their apoptosis-inducing activity. It has been recently reported that many of anticancer agents and radiation therapy induced autophagic cell death especially in human malignant glioblastoma cell lines. However, it has not been clear which type of cell death is induced by MMC in human oral SCC cell lines.Materials and MethodsHuman promyelocytic leukemia HL-60 cells were cultured in RPMI 1640 medium supplemented with 10% FBS in a humidified 5% CO2 atmosphere. Human oral normal cells (gingival fibrobast HGF, pulp cell HPC, periodontal ligament fibroblast HPLF), human oral squamous cell carcinoma (HSC-2, HSC-3, HSC-4, Ca9-22, NA), human malignant glioblastoma cell lines (T98G, U87MG) were cultured in DMEM medium supplemented with 10% heat-inactivated FBS. We used three antitumor antibiotics, MMC (Merck KGaA, Darmstadt, Germany), bleomycin (BLM) (Sigma Chem Co., St Louis, MO, USA) and PEP (Nippon Kayaku Co., Ltd., Tokyo, Japan).Method of Data AnalysisAssay for cytotoxic activity: The relative viable cell number of adherent cells was then determined by the MTT method. The viable cell number of HL-60 cells in suspension culture was determined by cell count with a hemocytometer after staining with 0.15% trypan blue.Analysis of apoptosis markerAnnexine V staining, caspase activation, DNA fragmentation, Western blot analysis, measurement of intracellular concentration of polyamine. Analysis of autophagy marker: Acridine orange staining.ResultsAmong these three compounds, MMC showed the highest cytotoxicity and tumor-specificity. However, there was considerable variation of drug-sensitivity among six cancer cell lines. MMC induced various apoptosis markers such as positive staining with annexine V, internucleosomal DNA fragmentation, caspase-3, −8, −9 activation and reduced expression of Bcl-2 protein in HSC-2 cells. MMC, however, did not induce most of these markers except the positive staining with annexin V in HSC-4 cells. Both of HSC-2 and HSC-4 cells did not produce an increase in the acid organelle (detected by acridine orange staining). On the other hand, MMC induced the formation of acid organelle, without induction of any of apoptosis markers in the human malignant glioblastoma cell lines (T98G, U87MG). MMC induced the decline of intracellular concentration of putrescine and spermidine in HSC-2 cells, and also induced the decline of that of putrescine in HSC-4 cells, but not in T98G, and U87MG cells.ConclusionWe investigated the induction of apoptosis in HSC-2 and HSC-4 cells. The present study suggests the diversity of drug sensitivity in hum an oral SCC cell lines, and that the type of cell death may depend on the drug sensitivity of the cells. Statement of the ProblemPeplomycin (PEP) and Mitomycin C (MMC) are used very often as neo-adjuvant chemotherapy before radical surgery. In contrast to hundreds of reports about the effect of MMC in vitro, there is little comparative study of cytotoxicity against bothhuman oral squamous cell carcinoma (SCC) cell lines and human oral normal cell lines. In the past, many of such studies have dealt with their apoptosis-inducing activity. It has been recently reported that many of anticancer agents and radiation therapy induced autophagic cell death especially in human malignant glioblastoma cell lines. However, it has not been clear which type of cell death is induced by MMC in human oral SCC cell lines. Peplomycin (PEP) and Mitomycin C (MMC) are used very often as neo-adjuvant chemotherapy before radical surgery. In contrast to hundreds of reports about the effect of MMC in vitro, there is little comparative study of cytotoxicity against bothhuman oral squamous cell carcinoma (SCC) cell lines and human oral normal cell lines. In the past, many of such studies have dealt with their apoptosis-inducing activity. It has been recently reported that many of anticancer agents and radiation therapy induced autophagic cell death especially in human malignant glioblastoma cell lines. However, it has not been clear which type of cell death is induced by MMC in human oral SCC cell lines. Materials and MethodsHuman promyelocytic leukemia HL-60 cells were cultured in RPMI 1640 medium supplemented with 10% FBS in a humidified 5% CO2 atmosphere. Human oral normal cells (gingival fibrobast HGF, pulp cell HPC, periodontal ligament fibroblast HPLF), human oral squamous cell carcinoma (HSC-2, HSC-3, HSC-4, Ca9-22, NA), human malignant glioblastoma cell lines (T98G, U87MG) were cultured in DMEM medium supplemented with 10% heat-inactivated FBS. We used three antitumor antibiotics, MMC (Merck KGaA, Darmstadt, Germany), bleomycin (BLM) (Sigma Chem Co., St Louis, MO, USA) and PEP (Nippon Kayaku Co., Ltd., Tokyo, Japan). Human promyelocytic leukemia HL-60 cells were cultured in RPMI 1640 medium supplemented with 10% FBS in a humidified 5% CO2 atmosphere. Human oral normal cells (gingival fibrobast HGF, pulp cell HPC, periodontal ligament fibroblast HPLF), human oral squamous cell carcinoma (HSC-2, HSC-3, HSC-4, Ca9-22, NA), human malignant glioblastoma cell lines (T98G, U87MG) were cultured in DMEM medium supplemented with 10% heat-inactivated FBS. We used three antitumor antibiotics, MMC (Merck KGaA, Darmstadt, Germany), bleomycin (BLM) (Sigma Chem Co., St Louis, MO, USA) and PEP (Nippon Kayaku Co., Ltd., Tokyo, Japan). Method of Data AnalysisAssay for cytotoxic activity: The relative viable cell number of adherent cells was then determined by the MTT method. The viable cell number of HL-60 cells in suspension culture was determined by cell count with a hemocytometer after staining with 0.15% trypan blue. Assay for cytotoxic activity: The relative viable cell number of adherent cells was then determined by the MTT method. The viable cell number of HL-60 cells in suspension culture was determined by cell count with a hemocytometer after staining with 0.15% trypan blue. Analysis of apoptosis markerAnnexine V staining, caspase activation, DNA fragmentation, Western blot analysis, measurement of intracellular concentration of polyamine. Analysis of autophagy marker: Acridine orange staining. Annexine V staining, caspase activation, DNA fragmentation, Western blot analysis, measurement of intracellular concentration of polyamine. Analysis of autophagy marker: Acridine orange staining. ResultsAmong these three compounds, MMC showed the highest cytotoxicity and tumor-specificity. However, there was considerable variation of drug-sensitivity among six cancer cell lines. MMC induced various apoptosis markers such as positive staining with annexine V, internucleosomal DNA fragmentation, caspase-3, −8, −9 activation and reduced expression of Bcl-2 protein in HSC-2 cells. MMC, however, did not induce most of these markers except the positive staining with annexin V in HSC-4 cells. Both of HSC-2 and HSC-4 cells did not produce an increase in the acid organelle (detected by acridine orange staining). On the other hand, MMC induced the formation of acid organelle, without induction of any of apoptosis markers in the human malignant glioblastoma cell lines (T98G, U87MG). MMC induced the decline of intracellular concentration of putrescine and spermidine in HSC-2 cells, and also induced the decline of that of putrescine in HSC-4 cells, but not in T98G, and U87MG cells. Among these three compounds, MMC showed the highest cytotoxicity and tumor-specificity. However, there was considerable variation of drug-sensitivity among six cancer cell lines. MMC induced various apoptosis markers such as positive staining with annexine V, internucleosomal DNA fragmentation, caspase-3, −8, −9 activation and reduced expression of Bcl-2 protein in HSC-2 cells. MMC, however, did not induce most of these markers except the positive staining with annexin V in HSC-4 cells. Both of HSC-2 and HSC-4 cells did not produce an increase in the acid organelle (detected by acridine orange staining). On the other hand, MMC induced the formation of acid organelle, without induction of any of apoptosis markers in the human malignant glioblastoma cell lines (T98G, U87MG). MMC induced the decline of intracellular concentration of putrescine and spermidine in HSC-2 cells, and also induced the decline of that of putrescine in HSC-4 cells, but not in T98G, and U87MG cells. ConclusionWe investigated the induction of apoptosis in HSC-2 and HSC-4 cells. The present study suggests the diversity of drug sensitivity in hum an oral SCC cell lines, and that the type of cell death may depend on the drug sensitivity of the cells. We investigated the induction of apoptosis in HSC-2 and HSC-4 cells. The present study suggests the diversity of drug sensitivity in hum an oral SCC cell lines, and that the type of cell death may depend on the drug sensitivity of the cells.