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BACKGROUND: Chinese hamster ovary cell line has been used routinely as a bioproduction factory of numerous biopharmaceuticals. So far, various engineering strategies have been recruited to improve the production efficiency of this cell line such as apoptosis engineering. Previously, it is reported that the caspase-7 deficiency in CHO cells reduces the cell proliferation rate. But the effect of this reduction on the CHO cell productivity remained unclear. Hence, in the study at hand the effect of caspase-7 deficiency was assessed on the cell growth, viability and protein expression. In addition, the enzymatic activity of caspase-3 was investigated in the absence of caspase-7. RESULTS: Findings showed that in the absence of caspase-7, both cell growth and cell viability were decreased. Cell cycle analysis illustrated that the CHO knockout (CHO-KO) cells experienced a cell cycle arrest in G2/M phase. This cell cycle arrest resulted in a 1.7-fold increase in the expression of luciferase in CHO-KO cells compared to parenteral cells. Furthermore, in the apoptotic situation the enzymatic activity of caspase-3 in CHO-KO cells was approximately 3 times more than CHO-K1 cells. CONCLUSIONS: These findings represented that; however, caspase-7 deficiency reduces the cell proliferation rate but the resulted cell cycle arrest leads to the enhancement of recombinant protein expression. Moreover, increasing in the caspase-3 enzymatic activity compensates the absence of caspase-7 in the caspase cascade of apoptosis.
Subject(s)
Animals , Recombinant Proteins/biosynthesis , CHO Cells , Caspase 7/genetics , Cell Cycle Checkpoints , Recombinant Proteins/genetics , Cell Division , Cricetulus , Cricetinae , Gene Knockout TechniquesABSTRACT
BACKGROUND: Chinese hamster ovary (CHO) cells are the most commonly used mammalian host cell In the commercial-scale production of biopharmaceutical proteins. Modification of genes involved in apoptosis may improve the productivity of CHO cells. Executive caspases, including caspases 3 and 7, play critical roles in apoptosis. The effects of the ablation of the caspase 7 gene on proliferation and viability of CHO cells remains unknown. In this study, we applied clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) to target caspase 7 gene of CHO K1 cell via all in one and homology targeted integration strategies. Consequently, the effect of caspase 7 deficiency on cell proliferation, viability, and apoptosis was studied by MTT assay and flow cytometry. RESULTS: Findings of gel electrophoresis, western blotting, and sequencing confirmed the caspase 7 gene silencing in CHO cells (CHO-KO). Proliferation assay revealed that caspase 7 deficiency in CHO cells resulted in the reduction of proliferation in various CHO-KO clones. Besides, the disruption of caspase 7 had negative effects on cell viability in exposure with NaBu which confirmed by MTT assay. Results of flow cytometry using Anexin V/PI demonstrated that Nabu treatment (11 mM) declined the percentage of live CHO-K1 and CHO-KO cells to 70.3% and 5.79%. These results verified that the CHO-K1 cells were more resistant to apoptosis than CHO-KO, however most of CHO-KO cells undergone early apoptosis (91.9%) which seems to be a fascinating finding. CONCLUSION: These results reveal that caspase 7 may be involved in the cell cycle progression of CHO cells. Furthermore, it seems that targeting caspase 7 is not the ideal route as it had previously been imagined within the prevention of apoptosis but the relation between caspase 7 deficiency, cell cycle arrest, and the occurrence of early apoptosis will require more investigation.
Subject(s)
Animals , Cell Survival , Apoptosis , Cell Proliferation , Caspase 7/deficiency , Cricetulus , Cricetinae , CHO Cells , Caspase 7/geneticsABSTRACT
Objective To discuss the expression of caspase-7 (CASP7) and its prognostic role in colon cancer. Methods CASP7 expression data was retrieved from Ualcan database, and the survival data of CASP7 down-regulated colon cancer patients was retrieved from Ualcan database and Oncolnc database. Results In colon cancer, the expression of CASP7 was down-regulated and indicated a relatively worse prognosis. The expression of CASP7 was correlated with KRAS and TP53I3. Conclusions CASP7 is down-regulated in colon cancer and indicates a worse prognosis.
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Aim To study the inhibitory effect of ex-opolysaccharide from Rhizopus nigricans ( EPS) combined with oxaliplatin on colon cancer in rats and its mechanism. Methods Colon cancer model in rats was established by subcutaneous injection of 1,2-dime-thylhydrazine ( DMH). The experimental rats were randomly divided into five groups: Control group, model group, EPS group (150 mg • kg-1 ) , oxaliplatin group (10 mg • kg-1 ) and EPS + oxaliplatin group. The his-topathological changes of colon tissues in rats were observed by HE staining. The expression levels of Sur-vivin, caspase-3 and caspase-7 proteins in colon tissues were detected by Western blot and immunohisto-chemistry. Results HE staining results showed that the damage degree of colon tissues could be significantly improved in treatment groups. Compared with model group, the expression of Survivin protein in treatment groups decreased significantly, and the expression of caspase-3 and caspase-7 proteins increased ( P < 0. 05). Conclusions Both EPS and oxaliplatin inhibit colon cancer in rats, and the synergistic effect is more remarkable. Its mechanism may be through inhibiting the expression of Survivin protein and increasing the expression of caspase-3 and caspase-7 proteins, thereby promoting the apoptosis of tumor cells and inhibiting the occurrence and development of tumors.
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Recent studies have reported that caspase 7 has an apoptotic and nonapoptotic function. However, the relationship between caspase 7 and spermatogenesis remains unknown. This study aimed to investigate the possible function of caspase 7 during normal and abnormal spermatogenesis. The cleaved form of caspase 7 was detected in testis tissues at different postpartum times (5-14 weeks) by qRT-PCR, Western blot and immunohistochemistry (IHC). Then, the mice models of spermatogenic dysfunction were obtained by busulfan (30 mg kg-1 to further evaluate the potential function and mechanism of caspase 7. qRT-PCR and Western blot results showed that caspase 7 expression was gradually elevated from 5 to 14 weeks, which was not connected with apoptosis. IHC results revealed that caspase 7 was mainly located in spermatogenic cells and Leydig cells. In addition, spermatogenic dysfunction induced by busulfan gradually enhanced the apoptosis and elevated the expression of caspase 3, caspase 6, and caspase 9, but decreased the expression of caspase 7 in spermatogenic cells. However, when spermatogenic cells were mostly disappeared at the fourth week after busulfan treatment, caspase 7 expression in Leydig cells was significantly increased and positively correlated with the expression of caspase 3, caspase 6, and caspase 9. Therefore, these results indicate that caspase 7 has a nonapoptic function that participates in normal spermatogenesis, but also displays apoptotic function in spermatogenic dysfunction.
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Objective To study the expression of cIAP and Caspase‐7 in endometrial adenocarcinoma tissue and its relation with prognosis .Methods Fifty‐five cases tissues of endometrial adenocarcinoma patients were detected by immunohistochemistry assay from the First Affiliated Hospital of Henan University of Science and Technology from 2009 to 2010 .Histological classifica‐tion ,pathological stage ,lymph node metastasis ,age and other parameters were collected ,the survival and overall survival of the tumor were followed up ,immunohistochemistry was used to observe the expression of cIAP and Caspase‐7 in paraffin sections of pathological specimens ,and the relationship between them and the parameters were analyzed .Results The expression level of cIAP and Caspase‐7 was related to the histological classification ,pathological stage and lymph node metastasis of endometrial carcinoma (P0 .05) .There was significant difference between the positive expression and the negative expression of cIAP and Caspase‐7(P<0 .05) .Conclusion The expression level of cIAP and Caspase‐7 in patients with endometrial carcinoma is correlated with tumor grade and overall survival ,or can be used as a prognostic indicator for endometrial cancer .
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Objective To study the effects of Liensinine on apoptosis of 5637 cells, and its mechanism thereof. Meth?ods CCK-8 method and the colony formation test were used to detect cell viabilities, and then inhibition rates were calcu?lated. Flow cytometry was used to detect the effects of Liensinine on apoptosis of 5637 cells. Western blot assay was used to detect Caspase-7 protein expression. Results CCK-8 assay and colony formation test indicated that Liensinine inhibited the cell proliferation significantly. Results of flow cytometry indicated that Liensinine induced early apoptosis of 5637 cells. Western blot assay showed that Liensinine improved the expression of Caspase-7 and enhanced the activation of Caspase-7 in 5637 cells. Conclusion Liensinine could inhibit the proliferation of 5637 cells, induce early apoptosis, which may be re?lated with the enhanced expression of Caspase-7 and its activation.
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Objective To study the inhibitory effect of arsenic trioxide(AS2O3)on the tumor growth of breast cancer line MDA-MB-435s implanted subcutaneously in nude mice and its mechanism.Methods BALB/C-nu/nu nude mice were subcutaneously injected with MDA-MB-435s breast cancer cells that are ER-positive,and treated with intraperitoneal injection of AS2O3 and DDP in different concentrations.The implanted tumor was weighed,and tumor inhibition rates were calculated.The expression of PTEN and Caspase-7 induced by AS2 03 were examined by immunohistochemical method.Results The growth of implanted tumor was markedly inhibited with DDP,low dose and high dose AS2O3,and the inhibitory rates were 48.68%,32.80%,66.67%respectively.The immunohisto chemical staining showed that the number of PTEN and Caspase-7 protein increased markedly(P＜0.05).Conclu sions AS2O3 inhibits the growth of human breast cancer cell implanted tumor.The molecular mechanism of AS2O3 on induction of apoptosis of breast cancer cells may be throush increasing the expression of PrrEN and Caspase-7(P ＜0.05).
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PURPOSE : Several lines of evidence have indicated that deregulation of apoptosis is involved in the mechanism of cancer development. Caspase-8 activation plays a central role in the initiation phase of apoptosis, while caspase-7 is one of the main execution phase caspases of apoptosis. The aim of this study was to explore the possibility that genetic alterations of the caspase-8 and caspase-7 genes are involved in the development of human non-small cell lung cancer (NSCLC). MATERIALS AND METHODS : We have analyzed the entire coding region of both the caspase-7 and caspase-8 genes to detect the somatic mutations in 100 NSCLCs by using polymerase chain reaction (PCR)- single strand conformation polymorphism (SSCP). RESULTS : The PCR-SSCP analysis detected no mutations in the entire coding regions of both the caspase-7 and caspase-8 genes in the NSCLCs. CONCLUSION : The data presented here suggests that both the caspase-7 and caspase-8 genes may not be somatically mutated in human NSCLCs