In Vivo And Vitro Studies On Effects Of Sodium Butyrate On Proliferation, Apoptosis And Telomerase Activity In Human Laryngeal Carcinoma Hep-2 Cells

Laryngeal carcinoma is one of common malignant neoplasms in head and neckcancer, accounting for 1%～1.2% of all malignancies and ranked the second among allthe cancers which derived from epithelial cells in head and neck. Recently, the morbilityand mortality of Laryngeal carcinoma are increasing and threatens people's healthseriously. The triditional therapy for laryngeal carcinoma, including surgical treatment,radiotherapy, chemotherapy and so on, has been improved and greatly raised the fiveyears survival rate of the patients in recent decades. But surgery often brings seriousdestruction of laryngeal function to the patients and often lead to mutilation;radiotherapy is only effective for the patients in early stage of laryngeal carcinoma andcan not take good effect on the most patients who are already in late stage at the time ofdiagnosis or in relapse or metastasis; and patients always can not tolerate chemotherapydue to the severe toxic reaction and side effects. So the application of these routinetherapy is constrained greatly. Then to seek novel,valid treatment methods with littlepoisonous side effect becomes a hot research subject of ENT researchers.In recent years, a lot of new anti-cancer drugs aiming directly to the multiple targetsfor cancer therapy have been developed with the further study on the mechanism oftumor occurrence. Many new anti-cancer drugs were being used in clinic, but there stillhave many problems needed to be solved and their clinical aapplication prospects arestill limited. Then many researchers turn to focus on telomerase inhibitors and apoptosis inducers because of their comprehensive anti-cancer mechanism and mild toxicity.As a novle anti-cancer drug aiming at the targets for cancer therapy, histonedeacetylase inhibitors (HDIs) has attracted researchers' attension in recent years.Through regulating histones acetylation and deacetylation, HDIs can change thestructure of chromatin, lead to transcription of corresponding genes, further result inchanges of tumor cells' morphology and function, and eventually contribute toproliferation inhibition, induction of differentiation and apoptosis, and telomeraseactivity inhibition in tumor cells so that the tumor can be inhibited. Sodium butyrate(SB) is one atoxic short-chain fatty acid derived from dietary fiber in the course offermentation in colons and is a type of small molecular compound belonging to HDIs.Studies in vitro confirmed that SB can inhibit the proliferation of variety of tumor cellsand induce cell differentiation, apoptosis and inhibition of telomerase activity in thecells, and consequently results in comprehensive anti-cancer effects. Further studiesshowed that SB can inhibit the growth of the transplanted tumors in nude mice derivedfrom human prostate cancer, ovarian cancer and endometrial cancer without toxicreaction and side effect. These findings indicated that SB might produce marked effectsin cancer treatment as a promising apoptosis inducer and telomerase activity inhibitorwith high power and little toxicity. At present, the studies about the anti-cancer effectsof SB have extended to the tumors in alimentary system, respiratory system,genitourinary system, nervous system, bone tumors and so on, but the reports about theeffects of Sb on the tumors in head and neck are few and there has been no report aboutthe effects of SB on laryngeal carcinoma so far.With the development and popularity of cell culture technique, It becomes one ofthe most important selective way to search new anti-cancer agents through cancer celllines cultured in vitro. Hep-2, a human laryngeal epithelial carcinoma cell line, is widelyused in the studies in vitro about the diagnosis and treatment of laryngeal carcinomabecause it has most biological characteristics of human laryngeal squamous cellcarcinoma. In order to investigate the effects of SB on human laryngeal carcinoma,Hep-2 cells were cultured in vitro and used to observe the effects of SB on proliferation,apoptois and telomerase activity in the cells and to explore their mechanisms by using immunohistochemistry method, flow cytometry (FCM) and many kinds of molecularbiology tecniques in this study, which might provid new idea and approach for thetreatment of laryngeal carcinoma.The circumstance in vivo is very different from that in vitro. The findings in vitrocan not reflect exactly the effects of drugs in vivo. In this study, based on the experimentin vitro, Hep-2 cells were seeded subcutaneously into the nude mice to establish humanlaryngeal carcinoma xenograft modes which were divided into two groups and treatedwith SB and PBS respectively. The changes of the xenograft tumors and the toxicreaction of the mice were observed in order to identify the inhibitory effect of SB invivo on laryngeal carcinoma and the mild toxicity of SB to mice and investigate themechanisms of this effect, which might provide valuable experimental data for theexploitation and application of SB. The studies were divided into three parts listedbelow:First part: Effects of sodium butyrate on proliferation and apoptosis inhuman laryngeal carcinima Hep-2 cellsMethods1. The proliferative activity of the Hep-2 cells treated with SB at 0.625, 1.25, 2.5, 5or 10mmol/L for 12, 24, 36, 48, 60 or 72h was assessed respectively by methyl thiazolylterazolium (MTT) assay and morphological changes of the cells were observed byinverted microscope.2. The changes of Ultramicrostructure in the cells treated with SB at 2.5mmol/L for48h were observed by transmission electron microscope.3. The apoptosis index (AI) of the cells treated wih SB at 2.5mmol/L for 0, 24.48or 72h was detected by terminal deoxynucleotidyl transferase-mediated dUTPnick-end-labeling (TUNEL) assay respectively.4. The DNA of the cells treated with SB at 2.5mmol/L for 0, 24, 48 or 72h wasextracted respectively to show DNA fragmentation by agarose gel electrophoresis.5. The apoptosis rate and cell cycle distribution of the cells treated with SB at2.5mmol/L for 0, 24, 48 or 72h were respectively analyzed by FCM.6. The expression status of Ki-67 antigen and Survivin protein in the cells treated with SB at 2.5mmol/L for 0, 24, 48 or 72h was respectively detected by usingimmunohistochemistry method.7. Statistical analysis: The SPSS 13.0 statistical package program was used for allanalyses. Two-factors analysis of variance was employed in MTT assay followed byLSD analysis. One-factor analysis of variance was used in TUNEL assay, FCM, andimmunohistochemistry method followed by LSD analysis. The significant level wasα=0.05.Results1. The proliferative activity of the cells treated with SB was inhibited obviously.There were significant differences between different concentration groups (P＜0.01)and between different time groups (P＜0.05).The inhibitory rate of cell growth in thecells treated with SB at 10mmol/L for 72h reched to 70.43%. The morphologicalchanges of the cells were more obvious with the increasing concentration andprolonging action time of SB.2. The typical ultramicrostructure changes of the apoptotic cells, such asconcentration and margination of nuclear chromatin and apoptotic body, appeared in theHep-2 cells treated with SB at 2.5mmol/L for 48h under transmission electronmicroscope.3. DNA ladders presented in agarose gel at 24 or 48h after the treatment by SB at2.5mmol/L.4. The AI in the cells without treatment was 2.27±1.18, and it increased to13.63±1.90, 22.25±3.56, 33.50±2.75 respectively at 24,48,72h after the treatment by SBat 2.5mmol/L. There were significant differences between different time groups (P＜0.01).5. With the prolonging action time of SB, the apoptosis rate of the cells increasedgradually (There were significant differences between different time groups, P＜0.01);the proportion of the cells at G0/G1 stage incresed (P＜0.01); that at S stage decreased(P＜0.01); that at G2/M stage did not change obviously (P＞0.05) and the proliferationindex (PI) of the cells declined evidently (P＜0.01).6. After the treatment by SB, the expression levels of Ki-67 antigen and Survivin protein in Hep-2 cells were both down regulated obviously. With the prolonging actiontime of SB, the mean optical density (MOD) values of Ki-67 antigen and Survivinprotein were both decreased gradually and there were significant differences betweendifferent time groups (P＜0.01).Second part: Effects of sodium butyrate on telomerase activity and mRNAexpression of telomerase subunits in human laryngeal carcinoma Hep-2 cellsMethods1. The telomerase activity of the Hep-2 cells treated with SB at 2.5mmol/L for 0,24, 48 or 72h was examined respectively by using telomeric repeat amplificationprotocal (TRAP)-sliver staining method and the inhibitory effect of SB on telomeraseactivity in the cells was investigated through semiquantitative analysis.2. The mRNA expression status of telomerase subunits in the cells treated with SBat 2.5mmol/L for 0, 24, 48 or 72h was respectively detected by using reversetranscription -polymerase chain reaction (RT-PCR) method and the effect of SB on theexpression of telomerase subunits in the cells was investigated through semiquantitativeanalysis.3. Statistical analysis: The SPSS 13.0 statistical package program was used for allanalyses. One-factor analysis of variance was employed followed by LSD analysis. Thesignificant level wasα=0.05.Results1. After the treatment by SB, the telomerase activity of the Hep-2 cells declined. Incomparison with that in the cells without treatment, the telomerase activity in the cellstreated with SB at 2.5mmol/L for 24, 48, 72h decreased 25.36%, 61.11%, 86.68%respectively and there were significant differences between diferent time groups (P＜0.01).2. The mRNA expression level of human telomerase reverse transcriptase (hTERT),the key subunit of telomerase, in the cells treated with SB at 2.5 mmol/L for 24, 48, 72hdecreased 20.92%,55.09%,75.80% respectively in comparison with that in the cellswithout treatment and there were significant differences between diferent time groups (P＜0.01). No significant changes were observed in the mRNA expression of humantelomerase RNA(hTR) and human telomerase associated protein 1(hTP1) (P＞0.05), theother two subunits of telomerase, at different time points.Third part: Studies on inhibitory effect of sodium butyrate on humanlaryngeal carcinoma xenograft tumors in nude miceMethods1. Hep-2 cells cultured in vitro were injected subcutaneously into the right armpitsof the 12 BALB/C-nu/nu female nude mice, which were 4-6 weeks old, to establishhuman laryngeal carcinoma xenograft models. After the neoplasms formed, the micewere divided randomly into 2 groups, the experimental group and the control group, andthere were 6 mice in each group. The mice in experimental group were treated with SBat a dose of 2.2mg/g body mass per day, while those in control group were injected withPBS at the same volume as SB. The agents were injected into hypodermis around thetumors and the treatment lasted 4 weeks.2. The mental status, diet condition and action of the mice were observed every day.The size of the tumors and the body mass of the mice were measured every week todraw the growth curves of the tumors in different groups respectively and to evaluatethe toxicity of the agent. At the end of treatment, the mice were killed and the tumorswere stripped out of the mice and weighted. The inhibitory rate of tumor growth wascalculated. The histologic changes in the tumors and main organs of the mice, such asheart, liver, lung, spleen and kidney, were observed by light microscope.3. The ultramicrostructure changes in the xenograft tumors were observed bytransmission electron microscope.4. The AI of the xenograft tumors was detected by using TUNEL method.5. The expression status of Ki-67 antigen and Survivin protein in the xenografttumors was detected respectively by using immunohistochemistry method.6. The telomerase activity in the xenograft tumors was examined by usingsemiquantitative TRAP-sliver staining method.7. The mRNA exPression status of telomerase subunits in the xenograft tumors wassemiquantitatively analyzed by using RT-PCR method. 8. Statistical analysis: The SPSS 13.0 statistical package program was used for allanalyses. Independent-samples t test was employed. The significant level wasα=0.05.Results1. The size of the tumors in the experimental group was smaller than that in thecontrol group and this effect was more evident with the prolonging treatment period ofSB. At the end of treatment, the differences of tumor size and tumor mass betwwen twogroups were both significant (P＜0.05, P＜0.01). The inhibitory rate of tumor growthbased on volumes and based on mass reached 58.81% and 60.33% respectively.2. Light microscope showed that the tissue necrosis area in the tumors at theexperimental group was much larger than that at the control group; the atypia grade oftumor cells in the experimental group was lower than that in the control group and thedegree of tumor differentiation in the experimental group was higher slightly than thatin the control group. The morphologic characteristics of apoptotic cells, such askaryopycnosis, appeared in the experimental group under light microscope.3. The typical ultramicrostructure Changes, such as concentration and margination ofnuclear chromatin, appeared in the experimental group under transmission electronmicroscope.4. The number of apoptotic cells in the experimental group was much more thanthat in the control group. The AI in two groups was 3.10±0.37, 29.70±1.84respectively and there was significant difference between two groups(P＜0.01).5. The expression levels of Ki-67 antigen and Survivin protein in the experimentalgroup were both lower than that in the control group. The MOD values of Ki-67 antigenin two groups were 0.278±0.012, 0.186±0.100 respectively and there was significantdifference between two groups (P＜0.01). The MOD values of Survivin protein in twogroups were 0.149±0.007, 0.098±0.009 respectively and there was significantdifference between two groups too(P＜0.01).6. The telomerase activity in the experimental group was lower than that in thecontrol group. The gray scales of telomerase activity in two groups were 3858.00±412.67, 2018.83±269.84 respectively and there was significant difference between two groups(P＜0.01).7. The mRNA expression level of hTERT in the experimental group was muchlower than that in the control group. The relative optical density (ROD) values ofmRNA expression of hTERT in two groups were 0.60±0.05, 0.26±0.03 respectivelyand there was significant difference between two groups (P＜0.01). No significantdifferences were observed in the mRNA expression of hTR and hTP1 between twogroups(P＞0.05).8. In the course of treatment, no adverse effects of SB were observed in the mice.The values of final body weight(FBW)/initial body weight(IBW) in two groups wereboth more than 0.8 and it indicated that there was no toxic reaction in the mice. Noorganic changes and tumor metastasis appeared in the main organs of the mice in twogroups.ConclusionsFor the first time, the effects of SB on proliferation, apoptosis, cell cycle,expression of Ki-67 antigen and Survivin protein, telomerase activity and mRNAexpression of telomerase subunits in human laryngeal carcinoma Hep-2 cells andhuman laryngeal carcinoma xenograft tumors were investigated byimmunohistochemistry method, FCM and many kinds of molecular biologytechniques in this study. The conclusions listed below were drawed:1. SB could inhibit the proliferation of Hep-2 cells and this inhibitory effectshowed time and dose dependence.2. SB could induce cell apoptosis in Hep-2 cells and this effect showed timedependence.3. SB could down regulate the expression of Ki-67 antigen in Hep-2 cells in atime-dependent manner, which might be one of the mechanisms of SB to inhibit theproliferation of Hep-2 cells.4. SB could down regulate the expression of Survivin protein in Hep-2 cells in atime-dependent manner. The apoptosis induction effect of SB might be realized bydown regulating the expression of survivin protein.5. SB took part in regulating cell cycle in Hep-2 cells and blocked cell cycle at G0/G1 stage. The proliferation inhibition, apoptosis induction and telomerase activityinhibition effects of SB might be all related to the cell cycle arrest at G0/G1 stage.6. SB could inhibit the mRNA expression of hTERT and down regulated theactivity of telomerase in Hep-2 cells in a time-dependent manner, and had no effect onthe mRNA expression of hTR and hTP1. It was indicated that the inhibitory effect of SBon telomerase activity in Hep-2 cells might be realized by down regulation of hTERTmRNA expression.7. Human laryngeal carcinoma xenograft models were established successfully innude mice and it was confirmed that SB could inhibit the growth of xenograft tumors innude mice without poisonous side effects.8. Resembling the results of experiments in vitro, SB could down regulate theexpression of Ki-67 antigen and Survivin protein, induce cell apoptosis, and inhibit theactivity of telomerase by down regulating the mRNA expression of hTERT in laryngealcarcinoma xenograft tumors in nude mice.9. The inhibitory effects of SB on the growth of Hep-2 cells and human laryngealcarcinoma xenograft tumors were confirmed for the first time in this study. Themechanism of the effects might be related to cell cycle arrest at G0/G1 stage, downregulation of Ki-67 antigen and Survivin protein, induction of apoptosis, downregulation of hTERT mRNA expression and inhibition of telomerase activity. This studyprovided valuable experimental data for clinical researches about SB.