Effect On Apoptosis And Proliferation Of Pancreatic Carcinoma Cell BxPC-3 Taken By 5-FU And Sulfasalazine In Vivo And In Vitro

PrefaceApoptosis was active death process in cell regulated by gene due to being triggered from intra-,extra-cellular environment changes or death signal, severious unbalance of which had destructed action on organisms. It has been proved that tumorigenesis, progression were related to disbalanced apoptosis. Moribity of pancreatic adenicarcinoma accounted to 1%-2% in total malignant tumors, showing gradually increasing tendency. Now pancreatic carcinoma as an obstinate disease is hardly overcomed, showing 24.5%-43.4% in operative resection rate, and 19.6%-26.1% in 5-year survival ratio of postoperation. Beside surgical intervention, the only palliative option replys mostly on chemotherapy with gemcitabine or 5-fluorouracil with moderate success. Nowadays main cause of non-excellence of chemotherapeutic effect is that multiple drug resistance of pancreatic carcinoma caused by chemotherapeutic drugs is intrinsical drug resistance not by drugs induction. It has been believed that transcription factor NF-κB, as one of the most important anti-apoptosis genes, has been gained much attention as it was involved in tumorigenesis and progression, mainly because of its strong anti-apoptotic potential. The blockade of NF-κB activity was generally believed to downregulate a set of anti-apoptotic genes, such as Bcl-2, XIAP, leading to increased apoptosis. It has been believed recently that elevation of NF-κB activity plays a critical role in chemoresistance, invasion, metastasis, apoptosis of pancreatic cancer cell, elevation of NF-κB activity is one of the most important causes that leads to drug resistance caused by pancreatic carcinoma cell. One efficient agent for NF-κB inhibition is sulfasalazine that blocks the activity of the IKKs and that is clinically used for therapy of rheumatoid arthritis and ulcerative colitis. The use of NF-κB inhibitors may abolish this resistance. 5-FU was one of the most effective chemotherapeutics used for gastrointestinal tumors, which could induce apoptosis in some sensitive cancer cell lines by blocking the enzyme thymidylate synthase. The aim of the study was to abolish resistance of pancreatic adenocarcinoma cell to 5-FU by inhibiting NF-κB activity and to explore whether both 5-FU and sulfasalazine synergistically promoted inducing apoptosis of pancreatic carcinoma cell, inhibiting multiple drug resistance(MDR) gene expression and cell proliferation. In view of the purpose, some related experiment were undertaken in vivo and in vitro.ObjectiveThe aim of the study lied in abolishing chemotherapy resistance of 5-FU to pancreatic adenocarcinoma cell by inhibiting the activity of NF-κB and investigated whether anti-proliferative effect of cell line BxPC-3 treated by 5-FU and sulfasalazine was associated with inducing apoptosis of pancreatic carcinoma cell. Apoptosis-related genes (NF-κB(P65), Bcl-2, MDR1, and cyclinD1) in cell BxPC-3 treated by 5-FU, sulfasalazine alone or both at the level of mRNA and protein were measured by RT-PCR and western blot. It was to explore relationship between apoptosis-related genes above mentioned and interaction mechanisms and to provide some experimental evidences for further investigating internal association among apoptotic mechanism, the multiple drug resistance and cell proliferative cycle and for overcoming the multiple drug resistance of pancreatic adenocarcinoma.Methods1. Cell cultureCell line BxPC-3 was cultured in RPMI1640 medium supplemented with 10% fetal bovine serum, 2.0mmol/L glutamine, 100μ/ml penicillin, 100mg/L streptomycin in an incubating box including 5% CO₂ at 37℃.2. Growth inhibition ratios measured with MTT methodGrowth inhibition ratios of cell BxPC-3 treated by 5-FU (12.5, 25, 50, 100 mg/L),sulfasalazine (25, 50, 100, 200 mg/L) alone or both of them (100+200, 100+100, 50+200, 50+100mg/L) at different times were assayed by MTT method, negative control of correspondence time designed. Then absorpted optical density (value A) per hole was detected by enzyme-associated immune assay instrument at 570nm wavelength. Growth inhibition ratio was computed with the equation that is value A₁ of negative control - value A of treated group)/(value A₁ of negative control - value A₀ of blank control)×100%. The experiment above mentioned was repeated 3 times.3. Apoptotic rate of middle, advanced stage and cell cycle changes Of cell BxPC-3 measured with PI methodApoptotic rate of middle, advanced stage and cell cycle changes in the synchronized cell BxPC-3 treated by 5-FU (12.5, 25, 50, 100 mg/L), sulfasalazine (25, 50, 100, 200 mg/L) alone or both of them (100 + 200, 100 + 100, 50 + 200, 50 + 100mg/L) at different times were measured by PI staining, 3 negative controls designed were 0.09%NaCl, 0.2% DMSO, 0.09% NaCl+0.2% DMSO, respectively. Hypodyploid cell of sub-G₁ phase was apoptotic cell. Every sample was analyzed with CELL Quest software (Becton Dickson, USA) to gain the percentage of cell cycle and apoptotic ratio. PI(proliferative index) was calculated according to the following equation that was (S+G₂/M)/(G₀/G₁+S+G₂/M). 10,000 cells at every sample were measured. The above experiments were repeated 6 times.4. Early apoptotie rate detected with PI/Annexin-V double stainingApoptotic rate of initial stage and cell cycle changes in the synchronized cell BxPC-3 treated by 5-FU ( 100 mg/L) , sulfasalazine (200 mg/L) alone or both ( 100 +200 mg/L) at different times ( 12, 24, 36, 48h) were measured by PI staining, 3 negative controls designed were 0.09%NaCl, 0.2%DMSO, 0.09%NaCl + 0.2%DMSO, respectively. Cell washed by cold PBS solution 2 times, centrifugated 1000rpm 5 min 4℃, supematant discarded, cell resuspended in 200ul binding buffer, then 10ul annexin V-FITC and 5ul PI were added, mixed well-distribution gently, being away from light for 30min 4℃. Every sample added 300ul binding buffer should be measured in 1h. The above experiments were repeated 6 times.5. Expression of apoptotic related proteins measured by immunocytochemical methodExpression of protein NF-κB (P65) , Bcl-2, cyclinD1 in cell BxPC-3 treated by (100mg/L) 5-FU, (200mg/L) sulfasalazine alone or both for 24h with three negative controls designed, fixed with 95% alcohol for 24h, treated by 1% Triton-X-100 for 30min, combined with rabbit anti-human NF-κB (P65), Bcl-2, cyclinD1 antibody for 24h at 4℃, with biotin labeling goat anti-rabbit IgG 37℃for 10min, coloured with DAB, counterstained by hematoxylin, gradiently dehydrated and neutral gum mounting. These targets were analyzed semiquantitatively according to positive cell number and staining intensity.6. Morphologie changes in cell observed by phase-contrast microscopyMorphologic changes in cell BxPC-3 treated by (100mg/L) 5-FU, (200mg/L) sulfasalazine alone or both for 24h were observed including each corresponding control group.7. Ultrastructure changes in cell observed by transmission electron microscopyCell BxPC-3 was treated by (100mg/L) 5-FU, (200mg/L) sulfasalazine alone or both for 24h including each control group, washed 2 times with PBS, pro-fixed by 2.5% glutaraldehyde, post-fixed with 1% osmic acid, dehydrated by alcohol, following with resin embedment, ultramicrocuting, electron staining, observed by transmission electron microscopy.8. Fine structure on the surface of cell observed by scanning electron microscopyCell BxPC-3 was treated by (100mg/L) 5-FU, (200mg/L) sulfasalazine alone or both for 24h including each control group, washed 2 times with PBS, pro-fixed by 2.5% glutaraldehyde, stained by 0.3% argent nitrate, dried with liquid CO₂ after dehydration, embedded by spurting gold. 8 areas were taken in every sample and observed by the scanning electron microscopy at 10,000 times enlarged9. Subcutaneously heterotopic transplantation tumor modeling in experimental animal nude mouse and experimental groupingSex ratio between female and male was 3:1, mouse age was 4-6w, weight was (20±2)g in 66 BALB/c nu/nu nude mouses breeded on condition of SPF. Cultured cell line BxPC-3 on large scale was collected and concentration of unicell suspension was regulated to 5.0×10⁶/ml, 0.5ml suspension was inoculated in the shoulder and back of each nude mouse. Diameter of transplantation tumour accounted to 0.6-0.8cm for 2-3 months, then all nude mouses were divided into 11 groups that were combined groups (15+20, 15+10, 7.5+20, 7.5+10mg/kg), sulfasalazine (20, 10mg/kg), 5-FU (15, 7.5mg/kg) and each negative control that was control 1 ( NS:DMSO=1:1 mixed solution), control 2 (DMSO), control 3 ( NS ). According to the scheme above mentioned, drugs were injected in transplantation tumour, 1 time every other day for 4 week, nude was executed by dislocation of cervical vertebra. Tumour size was measured after turnout being taken out of nude. Tumour was put into EP tube and reserved at -70℃refrigerator.10. Apoptotic-related genes (Bcl-2, cyclinD1, Bax and NF-κB (P65)) at the levels of mRNA in pancreatic adenocarcinoma cell BxPC-3 and subcutaneously heterotopic transplantation tumor treated by 5-FU, sulfasalazine alone or both with RT-PCR method.Treating groups from transplantation tumor or cell BxPC-3 were collected including each control. Total RNA was extracted by RNA out kit and measured with ultra-violet spectroscopical luminosity meter, 1ul total RNA as a template was reversely transedpted and synthesized into cDNA according to RNA PCR Kit (AMV) Ver. 3.0 procedure from TakaRa company. Reaction system in RT-PCR was 10 ul. Inversely trancripted products were amplified by PCR meter, reaction system in which was 40 ul. Results of destined gene expression level were semiquantitatively analyzed with ultra-violet gel imaging analysis system, The above experiments were repeated 6 times.11. Apoptotic-related genes (Bcl-2, cyelinD1, Bax, MDR1 and NF-κB (P65)) at the levels of protein in pancreatic adenocarcinoma cell BxPC-3 and subcutaneously heterotopic transplantation tumor treated by 5-FU, sulfasalazine alone or both with western blotTreating groups from transplantation tumor or cell BxPC-3 were collected including each control, washed with cold PBS 3 times, put into cold lysate (50mmol/L Tris [PH 7.4], 150mmol/L NaCl, 2.0mmol/L EDTA, 1% NP-40), centrifugated 12000g for 15min at 4℃, 20μg lysate precipitated according to the above-mentioned method was added into sample buffer solution, boiled. Equal amount of protein was taken to undertake SDS-PAGE electrophoresis, being separated and transferred, blocked with evaporated skimmed milk, reacted with monoantibody (1:200) put for overnight at 4℃, rinsed with TTBS for 5min 2 times, reacted with second antibody (1: 200) for 2h, rinsed 2 times, colored with AP solution. The absorptive optical density in master tapes was evaluated by imaging analysis software Fluorchem V 2.0 to calculate the level of expressed protein above-mentioned, The above experiments were repeated 6 times.Results1. Cell growth inhibition ratio assessed by MTT methodGrowth inhibition effect on cell BxPC-3 treated by 5-FU and sulfasalazine at the different concentrations appeared in a time-, dose-dependent manner, while growth inhibition ratio of cell BxPC-3 treated by different concentrations of 5-FU gradually increased with time prolonged, while which of different concentrations of sulfasalazine showed decreasing trend at the lengthened time.2. Apoptotic rate in the middle, advanced stage and cell cycle changes in the cell BxPC-3 were detected by PI staining methodApoptotic rate of cell BxPC-3 induced by 200mg/L sulfasalazine, 100 mg/L 5-FU either or both for 12h was 2.68%, 6.59%, 10.52%, respectively compared with corresponding control groups that were 3.17%, 1.50%, 4.08% (t=2.33 (P＞0.05), 9.78, 17.56 (P＜0.01)) , it was added up to 7.63%, 40.43%, 64.69% for 48h in comparison with 29.20%, 5.61%, 12.02% of control groups (t=17.06, 33.66, 94.51 (P＜0.01)) . Apoptotic rates, S-phase cell proportion and proliferative index(PI) ascended in 5-FU (12.5, 25, 50, 75, 100mg/L) alone for 24h. However, apoptotic ratio slowly rised from 2.68% to 7.63%, along with G₀/G₁-phase proportion increasing from 35.13% to 54.32%, S-phase cell proportion descended from 45.37% to 16.67% and proliferative index gradually decreasing with increased concentrations of sulfasalazine for 24h. G₀/G₁-phase proportion of cell BxPC-3 in combined group was increased from 43.31% (12h) to 85.05% (48h) compared with control (t=7.93 (12h), 21.30(48h), P＜0.01), S-phase proportion desending from 11.63% (12h) to 4.47 (48h) compared with control (t=37.68 (12h), 8.60 (48h), P＜0.01).3. Early apoptotic rate of cell BxPC-3 treated by (100mg/L) 5-Fu, (200mg/L) sulfasalazine alone or both at different times with the detection of PI/Annexin-V double stainingwith time prolonged, early apoptotic rate of cell BxPC-3 treated by 5-FU alone gradually increased in contrast with concurrent negative control (t=5.41, 2.06, 13.38, 22.99, P＜0.01, except for P＞0.05 at 24h, while sulfasalazine treatment showed the trend of apoptotic rate firstly increase, later decrease, inferior to 5-FU treatment, compared with negative control(0.2%DMSO) (t=2.65, 4.53, 2.32, 3.78, P＜0.05). But early apoptotic rate of the combination took obviously increasing trend, higher than that of two single treatment, in comparison with negative control (0.09% NaCl +0.2% DMSO) (t=5.58, 17.35, 24.95, 33.67, P＜0.01). Early apoptotic rates of cell BxPC-3 treated by (100mg/L) 5-Fu, (200mg/L) sulfasalazine alone or both at 12,24,36,48h including negative control (0.09% NaCl +0.2% DMSO) were analyzed by multiple factor analysis of variance to show obvious interaction of both (F=52.23, 245.14, 277.34, 418.93, P=0.000, respectively).4. Result measured by immunocytochemical methodExpression of protein NF-κB (P65), Bcl-2, cyclin D1 treated by (100mg/L) 5-Fu, (200mg/L) sulfasalazine alone or both for 24h were evaluate, average absorption optical density (AOD) of which showed significant difference compared with each negative control; meanwhile the same target between each treated group has significant difference (P＜0.01). Average absorption optical density of Bcl-2 among comparison of (100mg/L) 5-Fu, (200mg/L) sulfasalazine alone or both including negative control (0.09% NaCl +0.2% DMSO) was analyzed by multiple factor analysis of variance to show obvious interaction of both (F=1216.57, P=0.000).5. Cellular morphological change observed by phase-contrast microscopyMost cells in the pyknosis and round forms started in the combined groups for 24h, negative control (0.09%NaCl+0.2%DMSO) showed a little cell becomed pyknosis and round ,cell body becoming a little swollen. More cell BxPC-3 treated by 5-FU (100mg/L) for 24h were in the pyknosis and round forms, negative control (0.09%NaCl) had basically no morphological changes. But a small amount of cell becomed pyknosis and round in the treating group of sulfasalazine (200mg/L) for 24h, a little swell of cell body was expressed in the negative control (0.2% DMSO).6. ultramicrostructure changes in cell BxPC-3 observed by transmission electron microscope.Chromatin of cell BxPC-3 treated by 5-FU and sulfasalazine accumulated below perinuclear membrane and apoptotic body formed, endocytoplasmic reticulum being abundant. Nuclear fragmentation appeared in (100mg/L) 5-FU group, cellular nmicrovillus enlargement and abundant cytoplasmic endoplasm in negative control. Karyopyknosis emerged in (200mg/L) sulfasalazine group, nuclear decomposed in negative control.7. Fine structure changes on the surface of cell BxPC-3 observed by scanning electron microscopeA large amount of villus ablated and shrinked in combined group in contrast with fraction of cell villus growing down in negative control. A great quantity of villus in 5-FU(100mg/L) group was atrophic, while villus enlargement emerged in negative control. Comparatively large villus appeared in cell BxPC-3 treated by 200mg/L sulfasalazine compared with control cell shrinkage and partly exfoliation.8. Changes at the levels of mRNA and protein of cell BxPC-3 treated by 5-FU, sulfaalazine alone or bothmRNA relative content of NF-κB (P65) in cell BxPC-3 treated by (100, 50mg/L) 5-FU, (100, 200mg/L) sulfasalazine for 24h was (0.51±0.06), (0.57±0.06), (0.19±0.03), (0.15±0.03), (0.24±0.04), (0.10±0.01), (0.40±0.06), (0.21±0.06), respectively, showing significant difference in comparison with each control (t=11.08, 9.12, 7.94, 9.96, 17.64, 34.45, 7.58, 12.82, P＜0.01). Expression of protein NF-κB (P65) in cell treated by (100, 50mg/L) 5-FU, (100, 200mg/L) sulfasalazine for 24h was (2980.60±153.77), (2209.87±143.04), (3294.90±61.57), (1451.47±68.19),(2615.60±237.03), (440.27±31.73), (2483.33±241.72), (1289.67±183.11), which was compared with each control to show significant difference t=7.99, 12.08, 23.79, 10.40, 6.49, 59.97, 7.27, 19.49, P＜0.01. Meanwhile, mRNA relative content and protein expression in Bcl-2, cyclin D1 were in contrast with each control to show significant difference. These targets above-mentioned were analyzed by multiple factor analysis of variance to show obvious interaction between the two.9. Changes in the levels of mRNA and protein of subcutaneously heterotopic transplantation tumor treated by 5-FU, sulfasalazine alone or bothmRNA relative content of NF-κB (P65) in subcutaneously heterotopic transplantation tumor treated by (7.5, 15mg/kg) 5-FU, (10, 20mg/kg) sulfasalazine was (0.42±0.07), (0.50±0.05), (0.35±0.06), (0.22±0.03), (0.26±0.05), (0.21±0.02), (0.15±0.02) and (0.09±0.03), respectively, showing significant difference in contrast with each control (t=19.16, 17.67, 0.71, 9.61, 13.58, 18.56, 21.78, 24.89, P＜0.01 except for t=0.71, P =0.48), which between every treating group was analyzed by multiple factor analysis of variance to show obvious interaction (F=35.04, P=0.00), in addition, protein NF-κB (P65) content was (2070.45±93.09), (2594.74±76.55), (3574.78±117.54), (1837.57±104.25), (1998.90±168.06), (1156.31±96.45), (2474.04±114.22), (669.92±98.75) in comparison with each control t=79.01, 67.49, 43.38, 0.71, 37.03, 64.84, 31.85, 76.09, P＜0.01, except for t=0.71, P=0.49. The three tagets including Bcl-2, cyclinD1 and MDR1 at the mRNA relative content and protein expression showed significant difference in contrast with each control (P＜0.01) and were analyzed by multiple factor analysis of variance to show obvious interaction between the both.Conclusion1. Sulfasalazine might inhibit cell BxPC-3 proliferation and enhance apoptotic effect induced by 5-FU. Combined group effect on cell BxPC-3 produced synergistically growth inhibitory action which was closely relevant to cell apoptosis of both co-induction and to that cell cycle was arrested in G₀/G₁-phase.2. Down-regulation of Bcl-2, cyclin D1, MDR1 and NF-κB (P65) at the levels of mRNA and proteins might be one of apoptotic effect mechanisms of cell BxPC-3 synergistically induced by 5-FU combined with sulfasalazine.3. Sulfasalazine synergistically enhanced apoptotic effect on pancreatic adenocarcinoma cell BxPC-3 from subcutaneously heterotopic transplantation tumor in nude mouse induced by 5-FU. Up-regulated level of Bax, down-regulated level of Bcl-2, cyclin D1, MDR1 and NF-κB (P65) might be one of apoptotic mechanisms in subcutaneously heterotopic transplantation tumor treated by 5-FU with sulfasalazine.