Sodium Phynylbutyrate Induced Differentiation And Regulated Sensitivity To Radiation And Chemical Therapy On Laryngeal Carcinoma Hep-2 Cells In Vitro

[BACKGROUD & OBJECTIVE]The inducing differentiation is a phenomenon that malignant tumor cells would turn "off biologically and reverting to a more "benign" phenotype at the presence of differentiation-inducing agents in vitro or in vivo. It's a cell reforming process and doesn't produce poison and ill reaction to normal cells. As an aromatic fatty acid, sodium phenylbutyrate (PB), which has been used for adjutant of the disorder of urea cycle, is being studied as a potential antitumour drug. As being shown in past studies, PB is a novel differentiation-inducing agent with broad spectrum. It can inhibit proliferation and induce differentiation to many erythroleukemia and solid tumor cells in laboratory models, such as Leukemia, lymphoma, malignant glioma, prostate carcinoma, colon carcinoma, cervical carcinoma and malignant melanoma. It makes PB promising candidate for antitumour therapy or combination with conventional therapies that PB achieved effective pharmacological concentration in vivo and didn't produce serious complications in no-tumor patients such as bone marrow depression, nephrotoxicity and peripheral nerve disease. At present, the mechanisms of PB inducing differentiation is not clear, but the study has tended to modulating theexpression of tumor cell genes. It had been proved that PB could directly inhibit histone deacetylase, enhance histone acetylation and resulting in DNA with a more open structure, which favors transcription. And by those functions PB could make the silence anti-oncogenes re-express and inhibit malignant phenotype related genes, which makes tumor cells develop to benign phenotype. Recently, the modulation of expression of cell cycle regulator gene became a new hot spot as cell cycle block induced by PB was detected widely in laboratory tumor models.Laryngeal carcinoma is a common malignant tumor of head and neck which main therapy methods are operation, radiotherapy and induction chemotherapy. The result of colligation treatment is not ideal to part of patients, because of the laryngeal impairment after operation, radiation resistance of some tumor, or the frequently complication and toxic reaction of induction chemotherapy. As it was reported that PB could reinforce tumors sensitivity to radiotherapy and chemotherapy, a new hope rise for better result of laryngeal carcinoma treatment.In the present studies, by studying the inducing effect of PB on laryngeal carcinoma cells Hep-2 in vitro, we detected the change of growth, differentiation and expression of cell cycle regulator gene after inducing by different concentration PB. Then we characterized the interactions between PB and radiation or agents in induction chemotherapy on Hep-2 cells in vitro. [METHODS]Laryngeal carcinoma cells Hep-2 was cultured in routine. The growth curves were drawn when Hep-2 cells treated with On K 2% 4mmol/L PB concentrations and the doubling times were calculated. Treating Hep-2 cells with different concentration of PB for several days, the form of cell morphology was observed with light microscope and transmission electron microscope. Then soft agar clone forming test, scratch test, MTT assay and flow cytometry were used to examine the changes of clone forming suppression rate, cell inhibited rate, half inhibited concentration (IC50), move ability, cell cycle and apoptosis rate. In order to evaluate the express of P16INK4\ p21WAf\ p27KIP1 and p53 genes induced by PB, RT-PCR and Western blotting were performed. At last, the plating clone forming test and MTT were used toexamine the growth inhibition of Hep-2 cells treated by the combination of PB with radiotherapy and 5-FU or CDDP in vitro. [RESULTS]1 Clone formation: The clone formation of control group was earlier, more and bigger relatively whereas the effective clone formation of PB treating groups decreased gradually following to the increase of PB concentration. For example, most cells treated with 4mmol/L PB could not form effective clone and the inhibited rate of clone formation achieved about 79.58%. There was significant difference between every PB concentration group and the control group except for lmmol/L PB group2 Growth curve: The growth curve of lmmol/L PB group was close to that of the control group. But the cell proliferation deceased apparently after treating with 2 or 4mmol/L PB. The treating effect of 4mmol/L PB was more significant that the cell amount was only 20% of the control group at the seventh day. The doubling time of the control group was 35.8 hours whereas that of 1, 2, 4mmol/L PB groups were 38.1,46.5 and 78.0 hours respectively.3 Cell morphology: Under control condition, polygon cells grew more rapidly and presented a cobblestone-like appearance o After treated for 4 days with 4mmol/L PB, the cell borders contracted and the shape of cells changed to fusiform with long cytoplasmic processes. The nucleus-plasm ratio descended while nucleolus become smaller. And the glycogen granules collecting in cytoplasm decreased and distributed stepless. Some cells changed like early phase of apoptosis and part of them shown apoptotic bodies.4 Cell mobility: The ability of Hep-2 cells moving to cover the scratch decreased after treating with lmmol/L PB for 6 days.5 Cell proliferation: Except for lmmol/L PB group, the cell-inhibited rate of every PB concentration group was high significantly than that of the control group (P<0.01). The maximum inhibited rate achieved to 90%. There was a positive correlation between the cell-inhibited rate and PB concentration (PR <0.01). The IC50 of PB for 3or 4 days was about 5.82 and 4.21mmol/L. Comparing the two time groups, the inhibition of same PB concentration were significantly different (P<0.01).6 Cell cycle and apoptosis: Treated with PB for 4 days, cells blocking Gl phase increased gradually following the increase of PB concentration comparing with control group (P<0.01) while cells in S or M phase decreased corresponding. The apoptotic rate of control group and 1, 2, 4mmol/L PB groups were 0.07%, 0.11%, 2.07% and 11.68% respectively.7 Expression of cell cycle regulating gene: The result of RT-PCR and Western blotting shown that normal Hep-2 cell expressed mRNA and protein of pl6INK4As p21WAI\ p27KIP1and p53. After treated with PB, mRNA and protein of pi6INIC4A descended a little (P>0.05), while the expression of p21WAF1, p27K1P1and p53 genes increased significantly in dose dependent(P<0.05).8 Effect of PB combined with radiation on Hep-2 cells: While PB pretreatment for 2 h, the clone survival fraction (SF) of every radiation dosage group decreased gradually following PB concentration increased. Comparing with simple radiation group, the effect of PB combined with radiation on clone SF was significant(F=3.549, P<0.01) . The proliferation of every therapeutic alliance group decreased comparing with simple radiation group.9 Effect of PB combined with induction chemotherapy agents on Hep-2 cells: Combined with PB, the cell-inhibited rate of 5-FU or CDDP was shown a significant dose-dependent increase (P <0.05) and the highest achieved to above 80%. The difference between PB concentration groups was significant (P <0.05) . The most increase of cell-inhibited rate was about 14.7% when 5-FU combined with PB whereas it was only 10.7% when CDDP combined with PB.[CONCLUSION]1 PB treatment caused a dose-dependent and time-dependent inhibition of the Laryngeal carcinoma Hep-2 cells proliferation.2 Hep-2 cells was induced by PB to miss non-contact inhibition, exit cell cycle, develop to more maturity cell morphology and decrease mobility. These changessuggested that the malignant phenotype of laryngeal carcinoma was altered in some degree, and confirmed that PB could induce differentiation on laryngeal carcinoma.3 Treating laryngeal carcinoma with higher PB concentration could induce apoptosis.4 PB could not induce expression of pl6INK4A that indicate pl6INK4A has no sense in PB blocking the cell cycle.5 The increasing of p21WAF1, p27Klpl and p53 expression after PB treatment suggested that both p21WAFI and p27KIPI maybe play roles in PB-inducing Gl/S cell cycle block. Since the time alteration of p21WAF1 and p53 was not detected, the relation of the two genes during cell cycle regulation of PB could not sure yet.6 Pretreated for 2 hours with low-dosage PB could enhance radiation sensitivity of Hep-2 Laryngeal carcinoma cell line in vitro in some degree.7 It was shown that PB could significantly enhance the cytotoxic effects of 5-FU or CDDP to laryngeal carcinoma cells Hep-2 in vitro and exert a synergistic effect when combined with 5-FU or CDDP. It suggested that PB could reinforce the effect of induction chemotherapy on Laryngeal carcinoma, reduce the dose of agents and decrease the occurrence of the complication and toxic reaction.