The Antitumor Effect Of Circularly Permuted TRAIL In Combination With 5-fluorouracil Against Human Colorectal Cancer

Colorectal cancer(CRC)is one of the most common digestive tract tumors with increasing morbidity and mortality year by year.It has become one of the main causes of cancer related deaths worldwide.In our country,the incidence and mortality of CRC show an obvious upward trend because of the development of social economy,the change of lifestyle,and the aging of population.Moreover,the age of onset has a tendency to be younger.5-Fluorouracil(5-FU)isoneofthemostcommonlyused chemotherapeutic drugs for metastatic CRC(mCRC)in the clinic.However,the inherent or acquired drug resistance hinders its antitumor effect.The prognosis of patients with mCRC is still poor,and the five-year survival rate of patients with mCRC is less than 10%.Therefore,it is of great importance to find effective strategies for the treatment of colorectal cancer.It is known that combined use of drugs is an effective way to overcome drug resistance.What is more,a deep understanding of specific molecular mechanisms of the combined effect is the key to the development of better combined therapies.Tumor necrosis factor related apoptosis inducing ligand(TRAIL)can induce apoptosis of a variety of human tumor and transformed cells,but has no obvious toxicity to normal tissues and cells.In addition,several studies showed that the effect of TRAIL-induced apoptosis could be enhanced and TRAIL resistance could be reversed when TRAIL was combined with cytotoxic drugs,radiation,and molecular targeted drugs in many malignant tumor cell lines.Circularly permuted TRAIL(CPT)or recombinant mutant human TRAIL(rmh TRAIL)is a mutant form of the native human TRAIL.Compared to wild-type TRAIL,CPT has significant improvements in terms of stability,solubility,and safety with prolonged antitumor activity.Studies have shown that CPT has a significant inhibitory effect on a variety of non-small cell lung cancer(NSCLC)cell lines and has no obvious toxic effect on human normal cells.When combined application of CPT and cisplatin,it showed a synergistic inhibition effect on NSCLC cells,and had no toxicity to normal human cells.Similar results were obtained from an in vivo experiment.Furthermore,a number of studies have shown that CPT has become a potential drug for the treatment of multiple myeloma.These all show that CPT has good prospects for antitumor therapy.However,the study of CPT has not yet been reported in colorectal cancer up to now.Therefore,in the present study,we investigated the effect of CPT or CPT combined with 5-FU on proliferation and apoptosis of colorectal cancer cell lines for the first time.We also explored the related molecular mechanisms of apoptosis induced by CPT in combination with 5-FU.We established nude mice model with CRC cell line HCT116 to observe the effect of CPT combined with 5-FU on tumor formation ability.Furthermore,we performed TUNEL assay to detect the apoptosis of human colorectal cancer transplanted tumor cells induced by CPT in combination with 5-FU.The main purpose of this study is to elucidate:(1)Whether CPT as a single agent can inhibit the cell proliferation and induce apoptosis of colorectal cancer cells.(2)Whether CPT in combination with 5-FU can enhance the antitumor effect and 5-FU can sensitize colorectal cancer cells to CPT-induced apoptosis.(3)The molecular mechanisms of antitumor effects of CPT combined with 5-FU against colon cancer cell lines.(4)The in vivo antitumor effect of CPT plus 5-FU.The results of the study would provide experimental evidence for future clinical trials of CPT in the treatment of colorectal cancer,and lay foundations for future clinical application of this combination regimen as a new treatment modality for advanced colorectal cancer.The study is divided into three parts listed below:Part One Effects of circularly permuted TRAIL in combination with 5-FU on the proliferation inhibition and apoptosis induction of colon cancer cellsObjective:To observe the effect of CPT,5-FU or both on the proliferation and apoptosis of colorectal cancer cell lines HCT116 and SW480in vitro.Methods:1.Cell proliferation inhibition analysis.Human colorectal cancer cell lines HCT116 and SW480 were seeded in96-well plates and left to adhere for 24h.Each cell line was divided into two groups:treated with different concentrations of 5-FU group and treated with different concentrations of CPT group.Both cell lines were then treated with different concentrations of 5-FU for 48h or with different concentrations of CPT for 12,24,or 48h.Afterwords,MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)was added to the medium to cultivate for 3h.Then,the absorbance value(OD value)was read using a microplate reader.The proliferation inhibition rate at each time point was calculated according to the formula,and then the half maximal inhibitory concentration(IC50)was obtained which was defined as the concentration of drug when cell inhibition rate reached at 50%.To investigate the antitumor effect of CPT in combination with 5-FU,CRC cell lines HCT116 and SW480 were seeded in 96-well plates and left to adhere for 24h.CRC cells HCT116 and SW480 were then treated with 5-FU(5μg/mL and 12.5μg/mL,respectively)and different concentrations of CPT alone or in combination for 48h.Subsequently,the cell inhibition rate was measured by the MTS assay as described above.2.Detection of the apoptosis rate of two colorectal cancer cells by flow cytometry.CRC cell lines HCT116 and SW480 were seeded in 6-well plates at a density of 5×10~5 cells per well.Each cell line was divided into six groups:control group,5-FU group,low dose CPT group,high dose CPT group,5-FU plus low dose CPT group,and 5-FU plus high dose CPT group.After adhereing overnight,5-FU and/or CPT were added to the cells to treat for 24h or 48h.Then the cells were collected and stained with Annexin V-FITC and propidium iodide(PI)together for 5 min at room temperature in the dark.Finally,the cells were analyzed using a FACS-Calibur flow cytometer.Results:1.5-FU or CPT as a single agent has obvious proliferation inhibition effect on two colorectal cancer cell lines.The results of MTS assay showed that 5-FU could inhibit the growth of both HCT116 and SW480 cells in a dose-dependent manner(P<0.001).HCT116 and SW480 CRC cells were treated with various concentrations of CPT for 12,24,or 48h.The results of MTS assay showed that CPT alone had an obvious inhibitory effect on the proliferation of CRC cells HCT116 and SW480 in a dose-and time-dependent manner(P<0.001).The IC50 values of CPT for HCT116 and SW480 cells at 48h were 8.49 ng/mL and 338.43 ng/m L,respectively.2.The inhibitory effect on cell proliferation is obviously enhanced when CPT combined with 5-FU against the two colorectal cancer cell lines.HCT116 and SW480 CRC cell lines were treated with CPT in combination with 5-FU for 48h.Then,the cell proliferation inhibition rates were determined.The results showed that compared to 5-FU or CPT alone,co-treatment with CPT and 5-FU significantly increased cell death in both cell lines.The differences were significant(P<0.001).3.5-FU can enhance the apoptosis induction effect of CPT in colorectal cancer cell lines.Cell apoptosis was detected by flow cytometry.The results showed that5-FU and CPT could induce cell apoptosis when used singly.when CPT combined with 5-FU,apoptosis significantly increased in both the HCT116and SW480 cell lines compared to that achieved by treatment with either agent singly(P<0.05).Moreover,the apoptosis rate at 48h was higher than that at24h in each experiment group(P<0.05).Summary:1.CPT as a single drug can inhibit the proliferation of CRC cells HCT116 and SW480 in a time-and concentration-dependent manner.5-FU singly can also inhibit the proliferation of these two cell lines,and the inhibitory effect is in a concentration-dependent manner.2.The inhibitory effect on proliferation of CRC cells HCT116 and SW480 is significantly enhanced by CPT combined 5-FU.3.CPT singly can induce apoptosis of CRC cells HCT116 and SW480,and the effect of apoptosis induction was obviously enhanced when CPT combined 5-FU.Part two Study of the mechanisms of apoptosis induced by circularly permuted TRAIL plus 5-FU in human colorectal cancer cellsObjective:To investigate the the mechanisms of apoptosis induced by co-treatment with CPT and 5-FU in human colorectal cancer cells.Methods:1.To detect the effect of CPT combined with 5-FU on the expression of related proteins in apoptotic signal pathway of human CRC cells by Western blot.CRC cell lines HCT116 and SW480 were seeded in 6-well plates at a density of 5×10~5 cells per well,allowed to adhere overnight.Each cell line was divided into four groups:control group,5-FU group,CPT group,and5-FU plus CPT group.Both cell lines were incubated with 5-FU or CPT alone or in combination for 48h.Then,the cells were washed with cold PBS and lysed on ice in RIPA lysis buffer(50 mM Tris-HCl pH 7.4,150 mM NaCl,1%NP-40,and 0.5%sodium deoxycholate).After the cell lysate protein was quantified using the bicinchoninic acid method.Cell lysates were then separated on 10%SDS-PAGE and transferred onto polyvinylidene difluoride(PVDF)membranes which then were blocked with 5%nonfat milk at room temperature for 2h,incubated with primary antibodies at 4°C overnight and followed by incubation with secondary antibodies at room temperature for 1.5h.The expression of related proteins were visualized using an enhanced chemiluminescence detection kit and analyzed using a chemiluminescent imaging system.2.Detection the effect of pan-caspase inhibitor z-VAD-FMK on apoptosis of the two CRC cell lines induced by CPT or CPT combined with5-FU by flow cytometry.HCT116 and SW480 CRC cells were treated with or without the pan-caspase inhibitor z-VAD-FMK 1h prior to incubation with CPT or CPT plus 5-FU for 48h.Then,the cells were collected and stained with Annexin V-FITC and PI together for 5 min at room temperature in the dark.Finally,the cells were analyzed using a FACS-Calibur flow cytometer.3.Detection of the effect of pan-caspase inhibitor z-VAD-FMK on the expression of related proteins in the signaling pathway of CRC cells apoptosis by Western blot.CRC cells HCT116 and SW480 were treated with or without the pan-caspase inhibitor z-VAD-FMK for 1h and then incubated with CPT or CPT in combination with 5-FU for 48h.Afterwards,the cells were lysed and proteins was collected.Cell lysates were then separated on 10%SDS-PAGE and transferred onto polyvinylidene difluoride(PVDF)membranes which then were blocked with 5%nonfat milk at room temperature for 2h,incubated with primary antibodies at 4°C overnight and followed by incubation with secondary antibodies at room temperature for 1.5h.The expression of related proteins was visualized using an enhanced chemiluminescence detection kit and analyzed using a chemiluminescent imaging system.Results:1.The effect of CPT combined with 5-FU on the expression of related proteins in the apoptotic signaling pathway.1.1 The apoptosis of CRC cell lines induced by CPT plus 5-FU is caspase-dependent.The results of Western blot analysis showed that CPT alone could activate caspase-3,caspase-8,and PARP in both HCT116 and SW480 CRC cells.Furthermore,the levels of cleaved caspase-3,caspase-8 and PARP significantly increased upon treatment with CPT plus 5-FU in these two cell lines.We found that the pro-form of caspase-9 decreased in both cell lines after treatment with CPT plus 5-FU,but the cleaved form of caspase-9 was not observed.1.2 5-FU can increase the expression of DRs in CRC cells.HCT116 and SW480 CRC cells were treated with different concentrations of 5-FU for 48h.Then,Western blot analysis was performed to detect the expression of DR4 and DR5 in HCT116 and SW480 CRC cells.Results showed that 5-FU elevated the expression of DR4 in both cell lines,but DR5 expression increased only in the HCT116 cells and not in the SW480cells.Compared with the control group,there was statistical significance(P<0.001).1.3 Combination of CPT and 5-FU downregulates the expression of the survival protein Bcl-XL.The results of Western blot analysis showed that the expression of Bcl-XL significantly decreased after treatment with CPT plus 5-FU in both HCT116 and SW480 cells.However,the expression of BAX,a pro-apoptotic protein belonging to the Bcl-2 family,was unchanged.Further,no obvious changes were noted in the expression of XIAP,a member of the IAP family.2.Pan-caspase inhibitor z-VAD-FMK can inhibit the apoptosis induced by CPT or CPT plus 5-FU.The results of the flow cytometric assay showed that z-VAD-FMK significantly inhibited the apoptosis of HCT116 and SW480 CRC cells induced by CPT or CPT plus 5-FU(16.3±0.4%vs 59.9±0.9%and 33.6±2.4%vs 90.8±0.8%in HCT116 cells;18.9±0.5%vs 43.7±0.2%and 29.7±0.2 vs 69.6±0.9%in SW480 cells)(P<0.001).3.Pan-caspase inhibitor z-VAD-FMK can block the transmission of apoptosis signaling in CRC cells.The results of Western blot analysis showed that the activation of caspase-3,caspase-8,and PARP was abrogated in both of the CPT and CPT plus 5-FU groups.We confirmed that the apoptosis induced by CPT or CPT combined with 5-FU was caspase-dependent in CRC cells.Summary:1.Apoptosis of CRC cells induced by CPT or CPT combined with 5-FU is caspase dependent.2.5-FU enhances apoptosis of CRC cells induced by CPT and increases the sensitivity of CRC cells to CPT by up-regulation DRs expression and down-regulation Bcl-XL expression.3.Pan-caspase inhibitor z-VAD-FMK significantly inhibits the apoptosis of CRC cells induced by CPT alone or CPT combined with 5-FU.4.Pan-caspase inhibitor z-VAD-FMK significantly decreases the expression of cleaved caspase-3,caspase-8 and PARP,and effectively blocks the cell signal transduction mediated by caspases.5.Caspases play a key role in the apoptosis of CRC cells induced by CPT or CPT combined with 5-FU.Part three Antitumor effect of circularly permuted TRAIL in combination with 5-FU in vivoObjective:To establish tumor-bearing nude mice model of human colorectal cancer,and to observe the antitumor effect of CPT combined with5-FU in vivo.Methods:1.To establish tumor bearing nude mice model of colorectal cancer HCT116 cells.The human CRC HCT116 cells at logarithmic growth phase were adjusted to 5×10~7 cells/mL.Then,CRC HCT116 cells were inoculated subcutaneously into the right armpit of six week old female BALB/C nude mice(6×10~6 cells per injection).When the tumor growed to 100-120mm~3,the mice were randomly divided into five groups(five nude mice in each group)and started to treat with medicine.The weight of the nude mice was measured every five days as well as the tumor diameter.The tumor volume was calculated according to the formula:Tumor volume(TV)=long diameter×short diameter~2×1/2.On the twenty-five day,the nude mice were killed.Then,the tumor tissue was stripped and weighed and photographed.The tumor tissues of each nude mice were divided into two parts,one was preserved in liquid nitrogen,and the other was soaked with 30%sucrose solution and made into frozen section.2.4’,6-diamidino-2-phenylindole(DAPI)and Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)assay.The sections were sliced with frozen section machine,and each piece was3μm.Then TUNEL/DAPI staining was performed according to the manufacturer′s instructions.Finally,the apoptosis of transplanted tumor cells in nude mice was observed by laser scanning confocal microscope and photographed.Results:1.The effect of CPT and/or 5-FU on the growth of tumor-bearing nude mice.During the whole experiment,the tumor-bearing nude mice were generally in good condition.There was no significant difference in body weight between nude mice in each group(P>0.05).The results showed that both CPT and 5-FU had no obvious adverse effect on tumor-bearing nude mice,either alone or in combination.2.Inhibitory effect of CPT in combination with 5-FU on the growth of CRC cells HCT116 xenograft in nude mice.CPT and 5-FU,as a single drug can inhibit tumor growth in nude mice,and there were significant differences compared with the control group(P<0.001).The inhibition of tumor growth in xenograft was more significant in combition groups,and the inhibitory effect in combination group of CPT(7.5mg/kg)plus 5-FU was the most significant.There was a significant difference between the combined groups and the other groups(P<0.001),and there was also a significant difference between the two combined groups(P<0.01).3.CPT combined with 5-FU ptomoted apoptosis of transplanted tumor cells in nude mice.The results of TUNEL/DAPI staining showed that CPT and 5-FU as a single agent could result in apoptosis of transplanted tumor cells in nude mice.The apoptosis in the combination groups was significantly higher than in other groups(P<0.001).The apoptosis induced by high concentration of CPT plus5-FU was the most significant.Summary:1.The combination of CPT and 5-FU showes a significant inhibitory effect on tumor growth of HCT116 tumor-bearing nude mice,and the combination of high concentration of CPT and 5-FU is more effective in inhibiting tumor growth.The tumor-bearing nude mice are well tolerated and have no obvious adverse reactions to this regimen.2.CPT combined with 5-FU promotes the apoptosis of tumor cells in HCT116 tumor-bearing nude mice.Conclusions:1.CPT as a single agent can inhibit the proliferation and induce apoptosis of colorectal cancer cells in vitro.CPT in combinatin with 5-FU can enhance the effect of proliferation inhibition and apoptosis induction of colorectal cancer cells in vitro.2.Apoptosis induced by CPT or CPT in combination with 5-FU is caspase-dependent.3.5-FU enhances apoptosis induced by CPT and increases the sensitivity of colorectal cancer cells to CPT by upregulating the expression of DRs and downregulating the expression of Bcl-XL.4.CPT alone can suppress the growth of transplanted tumors in nude mice in vivo,and the effect of tumor suppression is significantly enhanced when CPT combined with 5-FU.Furthermore,CPT in combination with 5-FU promotes the aopotosis of tumor cells in nude mice bearing human colorectal carcinoma.