CRAd.pEgrl-TRAIL-Smac联合放射对MDA-MB-231细胞杀伤效应的研究
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摘要
恶性肿瘤作为一种全身性疾病，其产生是由多基因、多途径及多因素参与逐步发展的复杂过程，单一疗法往往难以取得满意疗效，因此，合理地选择综合治疗的模式为有效地杀伤肿瘤细胞，彻底治愈癌症带来了希望。肿瘤基因-放射治疗的提出弥补了放射治疗与基因治疗各自的弊端，将二者有机地结合起来，发挥协同抗肿瘤作用，既增强了肿瘤组织对放射治疗的敏感性、提高外源基因靶向转移和杀伤肿瘤细胞的效率，同时也可以降低照射剂量，缓解射线对肿瘤周围正常组织的损伤。
本研究利用对肿瘤细胞具有靶向性的溶瘤腺病毒作为载体，利用Egr-1启动子可在辐射诱导下增强其下游目的基因表达的特性，以及TRAIL和Smac基因的协同促肿瘤细胞凋亡的作用，构建了携带TRAIL和Smac双基因的具有双重靶向的pAd.Egr1-TRAIL-Smac-hTERT-E1A-E1B-E1B55K的溶瘤腺病毒质粒，并在HEK293细胞中成功地将其包装成携带Egr-1启动子、TRAIL和Smac双基因的双重靶向溶瘤腺病毒CRAd.pEgr1-TRAIL-Smac，探讨在其联合X射线照射对MDA-MB-231细胞增殖及凋亡的影响，以及MDA-MB-231细胞凋亡途径中相关基因的mRNA和蛋白表达规律。实验结果表明，CRAd.pEgr1-TRAIL-Smac联合X射线照射对MDA-MB-231细胞具有明显的增殖抑制及其促凋亡作用，可使细胞中TRAIL、DR5、Smac、Cytc、caspase-8、-9和-3mRNA及蛋白表达增高。结果提示，CRAd.pEgr1-TRAIL-Smac联合放疗抑制MDA-MB-231细胞增殖和促进其凋亡的机制，涉及细胞S期和G2期阻滞及死亡受体途径与线粒体途径中TRAIL、DR5、Smac、Cytc、caspase-8、-9和-3等基因的相互作用。本研究结果为临床治疗肿瘤提供了重要的理论依据及实验基础。
Malignant tumor is one of diseases which mainly threaten human health. Traditional methods for treating tumors are surgery, radiotherapy, chemotherapy and biological therapy, but monotherapy often has its own limitations. Therefore, various means of combined therapy for tumors have become a hot research field. On the basis of the respective character of radiotherapy and gene therapy, Weichselbaum et al. proposed the presumption of tumor gene-radiotherapy, i.e. both regulatory sequence of ionizing radiation-induced gene and tumor-killing gene are transferred into tumor cells which are irradiated, which induce the expression of tumor-killing gene and form a synergism of inhibiting tumors by ionizing radiation and gene suppression.
Tumor necrosis factor related apoptosis inducing ligand (TRAIL) cloned by Wiley et al.(1995) is a member of tumor necrosis factor (TNF) superfamily. TRAIL can induce the apoptosis in tumor cells, transforming cells and virus-infecting cells, but not damage normal cells. TRAIL not only has the killing effect on tumor cells, but also induces the apoptosis of tumor cells when combined with radiotherapy, which bring about the synergistic effect of killing tumor cells and increase its sensitivity to irradiation. Some studies confirm that ionizing radiation can up-regulate the expressions of TRAIL and death receptors (DR4and DR5) and promote the apoptosis of tumor cells. TRAIL in combination with radiotherapy has significantly synergistic effect, and get a good curative effect on treating breast cancer in preclinical experiment and one and two stage clinical trials.
Smac is located in the mitochondria, with the stimulation of apoptosis signal, could release into the cytoplasm together with Cyt c, in which it can be in conjunction with IAPs, inhibit the anti-apoptotic activity of IAPs, and enhance apoptotic effect. Smac gene over-expression could enhance the sensitivity of tumor cells to radiotherapy, chemotherapy and TRAIL. Binding with death receptor, TRAIL can induce procaspase-8to be sheared into the caspase-8, and activate caspase-8-tBid-Bax cascade, which contribute to the mature Smac protein is rapidly released into the the cytoplasm to bind with XIAP, eliminating the inhibition of caspase-3, then activating caspase-3and-8, and resulting in tumor cells apoptosis via caspase cascade at last. TRAIL inducing apoptosis process is often hindered by IAPs, while Smac can lift its anti-apoptotic effects, and let TRAIL successfully playing its apoptosis-inducing effect. Therefore, the co-expression of TRAIL and Smac genes could enhance anti-tumor effect synergisticly.
In the present study, Egr-1promoter, TRAIL and Smac gene were inserted into a CRAD vector. The recombinant expression system had three functions:(1) The CRAD vector has a double targeting effect and disrupts to tumor cells;(2) Radiotherapy kills tumor cells and induces the transcription of Egr-1promoter, which triggers TRAIL and Smac;(3) TRAIL and Smac genes promote the apoptosis of tumor cells. Then, the recombinant multifunctional expression system CRAd.pEgr1-TRAIL-Smac combined with ionizing irradiation were used to kill human breast cancer cells. The integration of radiotherapy, virus therapy and gene therapy would provide a new approach to the combined therapy of malignant tumors.
1. Construction of conditionally replicative adenovirus vector
The TRAIL and Egr-1promotor genes were cloned from cDNA of human placenta tissue mRNA and T-Egrl as the templates. The conditionally replicative adenovirus vector of possessed double-targeting to tumor cells, pShuttle-Egrl-TRAIL-hTERT-E1A-ElBp-E1B55K, was constructed and packaged successfully. The recombinant adenovirus was amplimfied and identificated. The virus was amplimfied in HEK293cells and identificated by PCR and Western blot. The genes of Egr-1, TRAIL, hTERT, E1A-ElBp and E1B55K have been successfully restructured into the adenovirus CRAd.pEgrl-Trail. The recombinant adenovirus of CRAd.pEgr1-Trail was obtained successfully.
2. Experimental grouping
The ten groups in the experiment are as follows, control, CRAd.p, CRAd.pEgrl-TRAIL, CRAd.pEgr1-Smac, CRAd.pEgrl-TRAIL-Smac,2.0Gy, CRAd.p+2.0Gy, CRAd.pEgr1-TRAIL+2.0Gy, CRAd.pEgr1-Smac+2.0Gy and CRAd.pEgrl-TRAIL-Smac+2.0Gy.
3. Killing effects of recombinant adenovirus combined with X-ray irradiation on MDA-MB-231cells
The MDA-MB-231cells were irradiated by X-rays with different doses24h after treated with recombinant adenovirus. The results showed that, with the the irradiation doses enlarging, the inhibition of cell growth increased gradually. Among all the groups, the inhibition in the CRAd.pEgrl-TRAIL-Smac+2.0Gy group was more significantly. The CRAd. pEgrl-TRAIL-Smac combined with X-irradiation can reduce effectively the dose of irradiation alone.
4. Apoptosis in MDA-MB-231cells inducted significantly by recombinant adenovirus in combination with X-ray irradiation
Twenty four h after infected with recombinant adenovirus with5MOI, the cells were irradiated with2.0Gy, then cell apoptosis was detected by flow cytometer. The results showed that the cell apoptosis percentage increased6h after irradiation, and continued until48h later, did significantly in the CRAd.pEgrl-TRAIL-Smac+2.0Gy group (P<0.05or P<0.01). These findings indicated that CRAd.pEgr1-TRAIL-Smac in combination with irradiation enhanced cell apoptosis.
The cell cycle progress was also detected. The results showed that as compared with the control group, the percentage of S phase cells increased6h after irradiation, and that of G2+M phase cells increased significantly in the CRAd.pEgrl-TRAIL-Smac+2.0Gy group48h later. These results indicate that the cells treated with recombinant adenovirus in combination with X-ray irradiation caused the cell cycle arrests of S and G2phases, which could be related to the status of p53gene because mutate p53gene can't induce the G0/G1phase arrest.
5. Expressions of TRAIL, Smac, DR5, caspase-8,-9and-3mRNAs and proteins in MDA-MB-231cells induced significantly by recombinant adenovirus in combination with2.0Gy irradiation
To further elucidate the apoptotic signal transduction pathway in MDA-MB-231cells affected by recombinant adenovirus in combination with2.0Gy irradiation, real-time PCR, ELISA and Western blot analyses were performed to evaluate the expressions of TRAIL, Smac, DR5, caspase-8,-9and-3mRNAs and proteins in the cells treated with2.0Gy irradiation. The results from real-time PCR analysis showed that the expressions of TRAIL, Smac, DR5, caspase-8,-9and-3mRNAs elevated4h after treatment, and reached to the peak8h later, did significantly in the CRAd.pEgrl-TRAIL-Smac+2.0Gy group, then declined gragually. The results from Western blot analysis showed that the expressions of TRAIL, Smac, DR5, caspase-8,-9and-3proteins up-regulated6h after treatment with recombinant adenovirus in combination with2.0Gy irradiation, and reached to the peak12h later; while24h later TRAIL protein expression reached to the peak. At the same time-point, there were significant differences of these genes mRNA expressions between the control, CRAd.pEgrl-TRAIL-Smac+2.0Gy group and other groups (P<0.05or P<0.01). These results suggest that CRAd.pEgrl-TRAIL-Smac in combination with radiotherapy involved in enhancing the apoptosis of MDA-MB-231cells.
In conclusion, we constructed the pShuttle-TRAIL-Smac-hTERT-ElA (CR2)-E1Bp-E1B55K with double targeting to tumor cells, and packaged into the CRAD with Egr-1promoter, TRAIL and Smac genes. This recombinant adenovirus CRAd.pEgrl-TRAIL-Smac had the character of targeting proliferation and radiation-enhanced gene expression in tumor cells, and promoted significantly the apoptosis of MDA-MB-231cells as combined with ionizing radiation. These effects were related to the arrests of S and G2phases and the genes of apoptosis death receptor pathway and the mitochondrial pathway interactions. The results will provide the experiment base for clinical application of tumor gene-radiotherapy, and offer a new thought for combined therapy in tumors.
本研究利用对肿瘤细胞具有靶向性的溶瘤腺病毒作为载体，利用Egr-1启动子可在辐射诱导下增强其下游目的基因表达的特性，以及TRAIL和Smac基因的协同促肿瘤细胞凋亡的作用，构建了携带TRAIL和Smac双基因的具有双重靶向的pAd.Egr1-TRAIL-Smac-hTERT-E1A-E1B-E1B55K的溶瘤腺病毒质粒，并在HEK293细胞中成功地将其包装成携带Egr-1启动子、TRAIL和Smac双基因的双重靶向溶瘤腺病毒CRAd.pEgr1-TRAIL-Smac，探讨在其联合X射线照射对MDA-MB-231细胞增殖及凋亡的影响，以及MDA-MB-231细胞凋亡途径中相关基因的mRNA和蛋白表达规律。实验结果表明，CRAd.pEgr1-TRAIL-Smac联合X射线照射对MDA-MB-231细胞具有明显的增殖抑制及其促凋亡作用，可使细胞中TRAIL、DR5、Smac、Cytc、caspase-8、-9和-3mRNA及蛋白表达增高。结果提示，CRAd.pEgr1-TRAIL-Smac联合放疗抑制MDA-MB-231细胞增殖和促进其凋亡的机制，涉及细胞S期和G2期阻滞及死亡受体途径与线粒体途径中TRAIL、DR5、Smac、Cytc、caspase-8、-9和-3等基因的相互作用。本研究结果为临床治疗肿瘤提供了重要的理论依据及实验基础。
Malignant tumor is one of diseases which mainly threaten human health. Traditional methods for treating tumors are surgery, radiotherapy, chemotherapy and biological therapy, but monotherapy often has its own limitations. Therefore, various means of combined therapy for tumors have become a hot research field. On the basis of the respective character of radiotherapy and gene therapy, Weichselbaum et al. proposed the presumption of tumor gene-radiotherapy, i.e. both regulatory sequence of ionizing radiation-induced gene and tumor-killing gene are transferred into tumor cells which are irradiated, which induce the expression of tumor-killing gene and form a synergism of inhibiting tumors by ionizing radiation and gene suppression.
Tumor necrosis factor related apoptosis inducing ligand (TRAIL) cloned by Wiley et al.(1995) is a member of tumor necrosis factor (TNF) superfamily. TRAIL can induce the apoptosis in tumor cells, transforming cells and virus-infecting cells, but not damage normal cells. TRAIL not only has the killing effect on tumor cells, but also induces the apoptosis of tumor cells when combined with radiotherapy, which bring about the synergistic effect of killing tumor cells and increase its sensitivity to irradiation. Some studies confirm that ionizing radiation can up-regulate the expressions of TRAIL and death receptors (DR4and DR5) and promote the apoptosis of tumor cells. TRAIL in combination with radiotherapy has significantly synergistic effect, and get a good curative effect on treating breast cancer in preclinical experiment and one and two stage clinical trials.
Smac is located in the mitochondria, with the stimulation of apoptosis signal, could release into the cytoplasm together with Cyt c, in which it can be in conjunction with IAPs, inhibit the anti-apoptotic activity of IAPs, and enhance apoptotic effect. Smac gene over-expression could enhance the sensitivity of tumor cells to radiotherapy, chemotherapy and TRAIL. Binding with death receptor, TRAIL can induce procaspase-8to be sheared into the caspase-8, and activate caspase-8-tBid-Bax cascade, which contribute to the mature Smac protein is rapidly released into the the cytoplasm to bind with XIAP, eliminating the inhibition of caspase-3, then activating caspase-3and-8, and resulting in tumor cells apoptosis via caspase cascade at last. TRAIL inducing apoptosis process is often hindered by IAPs, while Smac can lift its anti-apoptotic effects, and let TRAIL successfully playing its apoptosis-inducing effect. Therefore, the co-expression of TRAIL and Smac genes could enhance anti-tumor effect synergisticly.
In the present study, Egr-1promoter, TRAIL and Smac gene were inserted into a CRAD vector. The recombinant expression system had three functions:(1) The CRAD vector has a double targeting effect and disrupts to tumor cells;(2) Radiotherapy kills tumor cells and induces the transcription of Egr-1promoter, which triggers TRAIL and Smac;(3) TRAIL and Smac genes promote the apoptosis of tumor cells. Then, the recombinant multifunctional expression system CRAd.pEgr1-TRAIL-Smac combined with ionizing irradiation were used to kill human breast cancer cells. The integration of radiotherapy, virus therapy and gene therapy would provide a new approach to the combined therapy of malignant tumors.
1. Construction of conditionally replicative adenovirus vector
The TRAIL and Egr-1promotor genes were cloned from cDNA of human placenta tissue mRNA and T-Egrl as the templates. The conditionally replicative adenovirus vector of possessed double-targeting to tumor cells, pShuttle-Egrl-TRAIL-hTERT-E1A-ElBp-E1B55K, was constructed and packaged successfully. The recombinant adenovirus was amplimfied and identificated. The virus was amplimfied in HEK293cells and identificated by PCR and Western blot. The genes of Egr-1, TRAIL, hTERT, E1A-ElBp and E1B55K have been successfully restructured into the adenovirus CRAd.pEgrl-Trail. The recombinant adenovirus of CRAd.pEgr1-Trail was obtained successfully.
2. Experimental grouping
The ten groups in the experiment are as follows, control, CRAd.p, CRAd.pEgrl-TRAIL, CRAd.pEgr1-Smac, CRAd.pEgrl-TRAIL-Smac,2.0Gy, CRAd.p+2.0Gy, CRAd.pEgr1-TRAIL+2.0Gy, CRAd.pEgr1-Smac+2.0Gy and CRAd.pEgrl-TRAIL-Smac+2.0Gy.
3. Killing effects of recombinant adenovirus combined with X-ray irradiation on MDA-MB-231cells
The MDA-MB-231cells were irradiated by X-rays with different doses24h after treated with recombinant adenovirus. The results showed that, with the the irradiation doses enlarging, the inhibition of cell growth increased gradually. Among all the groups, the inhibition in the CRAd.pEgrl-TRAIL-Smac+2.0Gy group was more significantly. The CRAd. pEgrl-TRAIL-Smac combined with X-irradiation can reduce effectively the dose of irradiation alone.
4. Apoptosis in MDA-MB-231cells inducted significantly by recombinant adenovirus in combination with X-ray irradiation
Twenty four h after infected with recombinant adenovirus with5MOI, the cells were irradiated with2.0Gy, then cell apoptosis was detected by flow cytometer. The results showed that the cell apoptosis percentage increased6h after irradiation, and continued until48h later, did significantly in the CRAd.pEgrl-TRAIL-Smac+2.0Gy group (P<0.05or P<0.01). These findings indicated that CRAd.pEgr1-TRAIL-Smac in combination with irradiation enhanced cell apoptosis.
The cell cycle progress was also detected. The results showed that as compared with the control group, the percentage of S phase cells increased6h after irradiation, and that of G2+M phase cells increased significantly in the CRAd.pEgrl-TRAIL-Smac+2.0Gy group48h later. These results indicate that the cells treated with recombinant adenovirus in combination with X-ray irradiation caused the cell cycle arrests of S and G2phases, which could be related to the status of p53gene because mutate p53gene can't induce the G0/G1phase arrest.
5. Expressions of TRAIL, Smac, DR5, caspase-8,-9and-3mRNAs and proteins in MDA-MB-231cells induced significantly by recombinant adenovirus in combination with2.0Gy irradiation
To further elucidate the apoptotic signal transduction pathway in MDA-MB-231cells affected by recombinant adenovirus in combination with2.0Gy irradiation, real-time PCR, ELISA and Western blot analyses were performed to evaluate the expressions of TRAIL, Smac, DR5, caspase-8,-9and-3mRNAs and proteins in the cells treated with2.0Gy irradiation. The results from real-time PCR analysis showed that the expressions of TRAIL, Smac, DR5, caspase-8,-9and-3mRNAs elevated4h after treatment, and reached to the peak8h later, did significantly in the CRAd.pEgrl-TRAIL-Smac+2.0Gy group, then declined gragually. The results from Western blot analysis showed that the expressions of TRAIL, Smac, DR5, caspase-8,-9and-3proteins up-regulated6h after treatment with recombinant adenovirus in combination with2.0Gy irradiation, and reached to the peak12h later; while24h later TRAIL protein expression reached to the peak. At the same time-point, there were significant differences of these genes mRNA expressions between the control, CRAd.pEgrl-TRAIL-Smac+2.0Gy group and other groups (P<0.05or P<0.01). These results suggest that CRAd.pEgrl-TRAIL-Smac in combination with radiotherapy involved in enhancing the apoptosis of MDA-MB-231cells.
In conclusion, we constructed the pShuttle-TRAIL-Smac-hTERT-ElA (CR2)-E1Bp-E1B55K with double targeting to tumor cells, and packaged into the CRAD with Egr-1promoter, TRAIL and Smac genes. This recombinant adenovirus CRAd.pEgrl-TRAIL-Smac had the character of targeting proliferation and radiation-enhanced gene expression in tumor cells, and promoted significantly the apoptosis of MDA-MB-231cells as combined with ionizing radiation. These effects were related to the arrests of S and G2phases and the genes of apoptosis death receptor pathway and the mitochondrial pathway interactions. The results will provide the experiment base for clinical application of tumor gene-radiotherapy, and offer a new thought for combined therapy in tumors.
引文
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