| Introduction:X linked inhibitor of apoptosis proteins (XIAP), which is expressed at high levels in many tumors, block apoptosis at the core of the apoptotic machinery by inhibiting caspases, confers radioresistance and in some groups of patients, associated with development and progression and dismal prognosis of malignancies. Laryngeal squamous carcinoma is one of the most common malignant neoplasms in the head and neck region, for which radiotherapy at early stage is very important to maintain the function of the Laryngeal. Cell death by apoptosis was implied to mediate therapy-induced cytotoxicity following irradiation. Defects in apoptosis may contribute to radiation resistance. To investigate the expression of XIAP in laryngeal squamous carcinomas and in cultured Hep-2 cells in vitro to observe the relationship between the apoptosis and variation of XIAP expression in Hep2 cells after irradiation, and to investigate the effect of XIAP up or down regulation by transfection with expression plasmid or antisense oligonucleotides (AS ODNs) on hep-2 cells apoptosis and radiotherapy sensitivity. We examined the expression of XIAP protein in laryngeal squamous carcinomas and in cultured Hep-2 cells followed byγray radiation. For further research, Upregulating or Downregulation of XIAP expression was achieved by expression vector plasmid (PGEX, PGEX-XIAP, PGEX-XIAP-mut, PGEX- XIAP?t), or G4 ASO transfected into hep-2 cells. The inhibition rate, apoptosis rate of hep-2 cells and XIAP expression in the levels of mRNA and protein were tested to find the relationship between XIAP expression and cell sensitivity to irradiation.Methods:1. Paraffin-embedded tissue specimens used for this study were obtained from 50patients of laryngeal squamous carcinomas. Using immunohistochemical staining for the paraffin sections (S-P methods), we examine the expression of XIAP protein in laryngeal squamous carcinomas and normal laryngeal tissues, and investigate the connection of the XIAP expression with the clinicopathological parameters.2. In vitro cultured Hep-2 cells were irradiated with 2, 4, 8 Gyγray and measured 48 hours after radiation to find the dose related index. Or cells were irradiated with4 Gyγray and measured 12, 24, 48 hours after radiation to find the time related index. We investigated the cell inhibition rate, cell apoptosis rate, expression of XIAP in mRNA level by RT-PCR and in protein level by flow cytometry (FCM).3. Upregulating XIAP expression was achieved by expression vector plasmid (PGEX, PGEX-XIAP, PGEX-XIAP-mut, PGEX-XIAP?t) transfected into hep-2 cells. 48hs after radiation the cell apoptosis rate, inhibition rate and XIAP expression in the levels of mRNA and protein were tested to find the relationship between XIAP over expression and cell sensitivity to irradiation. The different effect of the three XIAP gene on cell protection and apoptosis was analysed. 4. Downregulation of XIAP expression was achieved by G4 ASO transfected into cultured hep-2 cells followed by radiation 6 hours later. 48hs after radiation cells were observed under the fluorescence inverse phase microscope. At the same time the cell apoptosis rate, inhibition rate and XIAP expression in the level of mRNA and protein were tested.Results:1. The expression of XIAP protein was observed in the cytoplasm and nucleus. The color of positive staining was light or dark brown. There was more positive staining in laryngeal squamous carcinomas specimens than in normal laryngeal tissue, and the difference between them was significant. It showed significant correlation between XIAP expression and tumor clinical and pathological stage (p<0.05) by X2 test analysis.2. After irradiation, the the volume of adherent cells increased and the number of suspended cells increased accordingly. 48 hours following irradiation with 2, 4, 8 Gy, the rate of cell inhibition was 3.24%, 8.29%, 13.53%, and apoptosis rate was 3.27%, 5.33%, 8.22%, the fluorescent index (FI) of XIAP protein measured by FCM was 1.23, 1.46, 1.58 respectively. With the dosage of 4Gy, 12, 24, 48 hours following irradiation, the rate of apoptosis was 3.19%, 3.22%, 5.31%, with the corresponding FI of XIAP protein 1.11, 1.29, 1.57. Correlation analysis showed the cell inhibition rate is corresponded with cell apoptosis rate. Both of the two parameters were increased along with dose increment and prolonged time course following irradiation. The induction of XIAP expression happened quickly in 24 hours following radiation but the apoptosis rate did not increase at this time.On 48 hours, XIAP was no more increased accordingly the apoptosis rate was significantly higher. This two were negtively related.3. Transfection with vectors with different XIAP fragment, (PGEX, PGEX-XIAP, PGEX-XIAP mut, PGEX-XIAP ?t) was conducted to hep-2 cells. 24 hours later cells were irradiated with 4 Gyγray. 48 hours following irradiation the XIAP protein FI tested by FCM was 1.10, 1.60, 1.80, 1.87 respectivly. The cell survival rate was 92.6%, 99.6%, 95.4%, 93.4%, and the corresponding cell apoptosis rate was 11.82%, 5.72%, 10.36%, 9.71%. This indicate that transfection of expression vector plasmid caused significantly increase of XIAP expression and cell survival and decreased apoptosis. PGEX-XIAP transfection decreased apoptosis most remarkably with respect to the other expression vectors though PGEX-XIAP mut and PGEX-XIAP? t transfection caused higher XIAP protein level.4. G4 ASO was transfected into cultured hep-2 cells, the MTT resultes indicate that 48 hours after transfection cell inhibition rate was elevated along with increasing dose of ASO. Cells were radiated 24 hours after 800nmol·L-1 ASO transfection, and after another 24hours ASO down-regulated XIAP mRNA expression by 55%. The protein levels were lowered up to 48.45%. The cell inhibition rate was doubled. In contrast, the scrambled control oligotide caused minimal XIAP loss and less cell inhibit.The FI of XIAP is negatively related to cell apoptosis rate. (r=-0.88 p=0.00016)Conclusion:1. Expresses of XIAP is higher in laryngeal squamous carcinomas than in normal laryngeal tissues. The level of XIAP expression is associated with tumor clinical and pathological characteristics.2.γ-ray can inhibit the growth of Hep-2 cells. There was a positive relationship between the rate of cell inhibition and irradiation dosage. The death of Hep-2 cells after irradiation was due to apoptosis.At early stage after radiation the XIAP expression was upregulated but the apoptosis was not induced.after 24 hours the protein was not changed any more and the apoptosis rate was increased. The over expression of XIAP maybe one reason which caused the delayed apoptosis post radiation.3. Up-regulation of XIAP expression promote the cell survival with decreased apoptosis. Compared with XIAP, XIAP mut and XIAP? t show less effect in promoting cell survival after irradiation. Since loss or variation of RING region weakened the anti apoptosis ability of XIAP, the function of RING is to promote apoptosis in hep-2 cells induced by radiation.4. XIAP is a viable target for cancer therapy in human laryngeneal neoplasms. In cultured Hep-2 cells XIAP ASO can induce apoptosis and enhance the sensitivity of hep-2 cells to radiotherapy.
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