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1.
Article in Chinese | WPRIM | ID: wpr-923362

ABSTRACT

Objective@#To investigate the role of long non-coding RNA double homeobox A pseudogene 9 (DUXAP9) in head and neck squamous cell carcinoma (HNSCC) and to evaluate the expression level, molecular function and mechanism of DUXAP9 in HNSCC cells.@*Methods@#Differential expression of lncRNAs between normal and tumor tissues in HNSCC tissues were screened using lncRNA microarray, the expression level of DUXAP9 in HNSCC tissues and its relationship with prognosis were analyzed in the TCGA database. The expression levels of DUXAP9 in HNSCC tissues and cell lines were detected using qRT-PCR. The function in HNSCC cells after DUXAP9 silencing was evaluated using the CCK-8 assay, wound healing assay, Transwell migration assay and subcutaneous xenograft assay in nude mice. Changes in the transcription and translation of epithelial-mesenchymal transition (EMT)-related proteins in head and neck squamous cell carcinoma cells after DUXAP9 silencing were detected using qRT-PCR and Western blot.@*Results@#lncRNA microarray results showed that, compared to adjacent normal tissues, DUXAP9 was abnormally upregulated in HNSCC tissues. Analysis from TCGA database showed that, compared to HNSCC patients with low DUXAP9 expression, HNSCC patients with high DUXAP9 expression had poorer survival. The relative expression of DUXAP9 in HNSCC tissues and 4 HNSCC cell lines increased compared to paired adjacent normal tissues as detected using qRT-PCR. Silencing DUXAP9 significantly inhibited the proliferation, migration and expression of EMT-related genes in HNSCC cells. The silencing of DUXAP9 significantly inhibited subcutaneous tumorigenesis of the HNSCC cell line CAL27 in nude mice.@* Conclusion@#Silencing DUXAP9 significantly inhibited the proliferation of HNSCC cells and subcutaneous xenografts in nude mice. DUXAP9 may mediate the migration of head and neck squamous cell carcinoma cells via the EMT pathway.

2.
Acta Pharmaceutica Sinica ; (12): 786-792, 2021.
Article in Chinese | WPRIM | ID: wpr-876512

ABSTRACT

Sempervirine, a yohimbane-type alkaloid isolated from Gelsemium elegans, was found to significantly inhibit the cellular proliferation of U251 cells in vitro and in vivo in a dose-dependent manner. U251 cells were treated with 0-16 μmol·L-1 of sempervirine for 24, 48 or 72 h. An MTT assay and clone formation assay were used to investigate cell survival and clone formation. Hoechst staining and Annexin V-FITC/PI staining were used to measure cell apoptosis. The expression of PI3K, AKT, p-AKT, Bax, Bcl-2, caspase-3 and cleaved caspase-3 was determined by Western blot analysis. The antitumor effect of sempervirine in vivo was investigated by inoculating nude mice with U251 cells. All animal experiments were in strict accordance with the regulations of the Biomedical Ethics Committee of Fujian Medical University (Fujian, China). The results show that sempervirine significantly inhibits the proliferation and induces the apoptosis of U251 cells, promotes cleavage of caspase-3, down-regulates the protein expression of PI3K and Bcl-2/Bax, and inhibits phosphorylation of AKT in vitro. Intraperitoneal injection of 4 or 8 mg·kg-1·day-1 of sempervirine inhibits U251 cells tumor growth in the xenograft nude mice, and tumor weight decreased by 44.76% and 61.26%, respectively. Our study shows that sempervirine significantly inhibits the proliferation of U251 cells in vitro and in vivo, laying a foundation for further research and development of its anti-glioma effect.

3.
Article in Chinese | WPRIM | ID: wpr-876117

ABSTRACT

@#[Abstract] Objective: To explore the application value of human IL-15 transgenic NCG mice (NCG-hIL-15 mice) in preclinical evaluation of chimeric antigen receptor modified NK (CAR-NK) cell therapy for tumor treatment. Methods: qPCR and WB were performed to detect the expression of human IL-15 in the bone marrow and main organs (spleen, liver, lung, kidney and pancreas) of transgenic mice. After being transfused with human PBMC-derived NK (PB-NK) cells, the NCG-hIL-15 mice and control NCG mice were continuously monitored for the in vivo amplification of NK cells and the changes in body weight and survival time. Flow cytometry was used to detect the differential expressions of activated receptors and inhibitory receptors in amplified NK cells. WB was used to detect the expressions of perforin and granzyme-B. NCG-hIL-15 mice or NCG mice bearing MIAPaca-2 cell transplanted tumor were treated with anti-MUC1-CAR-NK cell reinfusion; then, the CAR-NK cell survival in different groups of mice was detected by Flow cytometry, and the survival time of tumor bearing mice was recorded and tumor growth was detected by in vivo imaging. Results: The results indicated that PB-NK cells could proliferate stably within 10 weeks in NCG-hIL-15 mice without obvious graft versus host diseases (GVHD) during the observation period. The in vivo-expanded human NK cells maintained the original expression patterns of various surface molecules, including KARs and KIRs. Compared with the NK cells in NCG mice, the NK cells in NCG-hIL-15 mice contained significantly higher amounts of granzyme-B and perforin (all P<0.05). CAR-NK cells showed significantly increased survival rate and stronger tumor-inhibitory effect in NCG-hIL-15 mice as compared with those in control NCG mice, resulting in significantly prolonged survival in NCG-hIL-15 mice (all P<0.01). Conclusion: NCG-hIL-15 mouse model has potential application value in preclinical trial and biological evaluation of NK cell-based immunotherapy.

4.
Article in Chinese | WPRIM | ID: wpr-942224

ABSTRACT

OBJECTIVE@#To establish an animal model with malignant tumor in the skull base-infratemporal region, and to explore the role of iodine staining technique in identifying tumor tissues with Micro-CT data.@*METHODS@#Sedation anesthesia was carried out on 12 BABL/c nude mice using inhaled isoflurane, and then WSU-HN6 cells that cultured and immortalized from human tongue squamous cell carcinoma were injected into the right infratemporal fossa via the submandibular area. The procedure was carried out under ultrasonographic guidance. The nude mice were sacrificed after 3 weeks observation. The head specimens were fixed and scanned by Micro-CT, and repeated scans were performed after staining with 3.75% compound iodine solution. Following decalcification in 20% EDTA for 2-4 weeks, the head specimens were embedded and sectioned. Hematoxylin and eosin staining and Pan-Keratin immunohistochemical staining were carried out. Bright-field microscopy and stereomicroscopy were used to visualize. The Micro-CT data were analyzed using iPlan software (Brainlab).@*RESULTS@#Non-traumatic ultrasonography was used to guide HN-6 cells injection and confirm skull-base tumor formation in all the animals. Ultrasonographic guidance reduced the risk of cervical vessel injury when transferring tumor cells into the skull base space. An obvious asymmetrical appearance was detected via ultrasonography 3 weeks after tumor cell injection. The Micro-CT analysis showed that the bone was obviously damaged on the right side of the skull base, but the soft tissue image was unrecognizable. After four days staining with compound iodine solution, the morphology of the tumor and surrounding soft tissue could be clearly identified. Hematoxylin and eosin staining showed the tumor formation of the right infratemporal fossa region accompanied by bone destruction. Human keratin immunohistochemical staining showed that the tumor tissue originated from human squamous cell carcinoma, and the polynuclear osteoclasts could be seen at the margin of the skull base bone resorption.@*CONCLUSION@#The animal model with malignant tumor in the skull base-infratemporal region could be successfully established via submandibular injection under ultrasound-guidance. Bone changes of the skull were easily observed on Micro-CT, but the tumor counter was not able to be distinguished from surrounding soft tissue. The 3.75% compound iodine staining of the head specimen could help discern the tumor and surrounding soft tissue in more details.


Subject(s)
Animals , Carcinoma, Squamous Cell/diagnostic imaging , Infratemporal Fossa , Iodine , Mice , Mice, Nude , Skull Base , Staining and Labeling , Tongue Neoplasms , X-Ray Microtomography
5.
Acta Anatomica Sinica ; (6): 32-39, 2020.
Article in Chinese | WPRIM | ID: wpr-844547

ABSTRACT

Objective To investigate the effect of interleukin( IL)-6 secreted by bone marrow stromal cells HS-5 on activity and apoptosis of human acute myeloid leukemia( AML) cells HL-60 and its possible mechanism. Methods HL-60 cells and HS-5 cells were cultured in vitro, and the co-culture system was established. Scanning electron microscope, ELISA, cell counting kit-8( CCK-8) , AnnexinV-FITC/PI, double-staining flow cytometry, Real-time PCR and Western blotting techniques were used to detect the changes of viability and apoptosis of HL-60 cells, respectively. HL-60 cells from different groups were inoculated into BALB/c nude mice to observe and record tumorigenesis. Results IL-6 secreted by bone marrow stromal cells HS-5 could enhance the viability and inhibit the apoptosis of HL-60 cells, and down-regulate the expression of pro-apoptotic gene Bax and and up-regulate the expression of anti-apoptotic gene Bcl-2 in HL-60 cells. HL-60 cells in co-culture group had the strongest tumorigenicity in BALB/c nude mice, while HL-60 cells alone had the weakest tumorigenicity. Conclusion Part of the mechanism by which bone marrow stromal cells HS-5 promote the proliferation of HL-60 cells and inhibit their apoptosis may be through the secretion of IL-6.

6.
Article in Chinese | WPRIM | ID: wpr-873018

ABSTRACT

Objective:To observe the effect of Zengmian Yiliu (ZMYL) formula and its effective components (PAWU) on the growth inhibition of ovarian cancer stem cell transplanted tumor in nude mice and the Notch signal receptor (Notch) / Notch signal ligand 1 (Jagged1) signal pathway in tumor tissue. Method:Ovarian cancer stem cells were cultured in serum-free suspension to establish the transplanted tumor model of ovarian cancer stem cells in nude mice, and then divided into model group, ZMYL group (36 g·kg-1), PAWU group (5.8 g·kg-1), cisplatin (DDP) group (2.5 g·kg-1), and PAWU (5.8 g·kg-1) + DDP group (2.5 mg·kg-1).After successful modeling, the drugs were given by gavage for 21 days.To observe the effect of Zengmian Yiliu decoction and its effective components on tumor weight in nude mice, the morphological changes of tumor cells were observed under light microscope, immunohistochemistry and real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)were used to detect the expressions of Notch 1, Jagged1, Hairy and enhancer of split 1 (Hes1) protein and mRNA in tumor tissues. Result:The tumor inhibition rates of ZMYL, PAWU, DDP and combination groups were 35.91%, 32.94%, 57.65% and 69.05%, respectively.Compared with the model group, the tumor weight of ZMYL group, PAWU group, DDP group and combination groups decreased significantly (P<0.05).Compared with PAWU group, the tumor weight of combination groups decreased significantly (P<0.05).Immunohistochemistry showed that compared with the model group, the positive expressions of Notch1, Jagged1 in ZMYL group, PAWU group, DDP group and combination groups were down-regulated (P<0.05),and the positive expressions of Hes1 in ZMYL group, DDP group and combination groups were down-regulated (P<0.05).Compared with combination groups, the positive expressions of Notch1, Jagged1 and Hes1 in ZMYL group, PAWU group, DDP group were up-regulated (P<0.05). Real time PCR showed that compared with the model group, the expressions of Notch1 mRNA in ZMYL group, PAWU group, DDP group and combination groups decreased significantly (P<0.05).Compared with model group, the expressions of Jagged1 and Hes1 mRNA in ZMYL group, PAWU group and combination groups decreased significantly (P<0.05).Compared with DDP group, the expressions of Notch1, Jagged1 and Hes1 mRNA in combination groups decreased significantly (P<0.05). Conclusion:The growth of ovarian cancer stem cells transplanted in nude mice can be inhibited by Zengmian Yiliu formula and its effective components.The effective components have a significant synergistic effect in the combination with cisplatin.Its mechanism is correlated to the inhibition of Notch/Jagged1 signaling pathway activation.

7.
Article in Chinese | WPRIM | ID: wpr-872915

ABSTRACT

Objective:To study the antitumor effect of Xihuangwan on A549 lung cancer nude mice in inflammatory microenvironment, and explore the effect of Xihuangwan on the expressions of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammatory bodies and their products in serum and tumor tissue of A549 lung cancer nude mice. Method:The lung cancer A549 cell model was established in nude mice with lung cancer, and the lung cancer A549 cell model was established in inflammatory microenvironment by adding lipopolysaccharide (LPS) + adenosine triphosphate (ATP) to the culture medium. After modeling, the rats were randomly divided into blank group (equal volume of normal saline), positive drug control group (MCC950 solution, 0.79 g·kg-1), and low, medium, high-dose Xihuangwan groups (0.39, 0.78, 1.95 g·kg-1). The rats were administered orally once a day for 21 days, and then sacrificed. The tumor tissues were stripped to measure the tumor body. The expressions of NLRP3, malondialdehyde(MDA), interleukin (IL)-1β, IL-18 and NLRP3 were detected by Western blot, and the levels of IL-1β, IL-18 and MDA were detected by enzyme linked immunosorbent assay(ELISA). Result:Compared with the blank group, the tumor inhibition rates in the positive drug control group and the low, medium and high dose Xihuangwan group were 39.21%, 31.72%, 42.24% and 55.68%. ELISA showed that the high-dose Xihuangwan group could significantly reduce the expressions of MDA, IL-1β and IL-18 in the serum of nude mice (P<0.05). Western blot showed that the high-dose Xihuangwan group could effectively reduce the protein expressions of MDA, IL-1β, IL-18 and NLRP3 in tumor of nude mice (P<0.05), the results of immunohistochemistry showed that the expression rate of NLRP3 in the tumor tissues of nude mice was reduced in the positive drug group and each dose of Xihuangwan group (P<0.05). Conclusion:Xihuangwan can inhibit the growth of tumor tissue of A549 cells bearing lung cancer in nude mice. The mechanism may be that it can inhibit the growth of tumor cells by inhibiting the expression of NLRP3 inflammatory bodies, IL-1β, IL-18, MDA tables, and then inhibiting the inflammatory microenvironment of tumor cells.

8.
Article in Chinese | WPRIM | ID: wpr-862241

ABSTRACT

@#[Abstract] Objective: To construct and verify the anti-tumor activity of chimeric antigen receptor (CAR) modified NK-92 cells (CAR-NK-92 cells) targeting prostate stem cell antigen (PSCA) in cervical cancer. Methods: Lentiviral vector expressing CAR targeting PSCA was constructed, and PSCA CAR-NK-92 cells were obtained by lentivirus transfection. The expression of PSCA in human cervical cancer cells was determined by Flow cytometry and Western blotting. The killing effect of PSCA CAR-NK-92 cells against cervical cancer cells was verified by co-incubation of effector and target cells in vitro, and the tumor inhibitory ability of PSCA CAR-NK-92 cells was verified with the nude mice xenograft model in vivo. Results: PSCA CAR-NK-92 cells were successfully constructed. PSCA was highly expressed in human cervical cancer Hela and MS751 cells (all P<0.01). In vitro co-incubation results showed that PSCA CAR-NK-92 cells could lyse PSCA+ cervical cancer transplanted tumor in a dose-dependent manner. In vivo anti-tumor data showed that PSCA CAR-NK-92 cells significantly inhibited the growth of cervical cancer cells compared with NK-92 cells transfected with vehicle vectors (P<0.01). In addition, PSCA CAR-NK-92 cells could effectively infiltrate tumor tissues and promote the secretion of anti-tumor cytokines TNF-α and IFN-γ (all P<0.01). Conclusion: The CAR-NK-92 targeting PSCA shows good anti-tumor effect on PSCA+ tumor cells both in vitro and in vivo, and has potential to be a therapeutic strategy for cervical cancer.

9.
Article in Chinese | WPRIM | ID: wpr-861516

ABSTRACT

Objective: To explore the effects of up- and down-regulation of circadian clock gene Bmal1 on the growth and radiation sensitivity of nasopharyngeal carcinoma after CNE1 xenograft in nude mice. Methods: We produced four groups of cells using lentiviral transfection: cells overexpressing CNE1 (CNE1OE), negative control in which there was no overexpression of CNE1 (OENC), cells with short hairpin RNAi (CNE1sh3), and RNAi negative cells (CNE1shNC). We investigated the expression of Bmal1 protein in the aforementioned groups with Western blot. After subcutaneously injecting the four groups of cells in nude mice, the size of the xenograft was measured. Subsequently, the xenografts were irradiated with 15 Gy at 6 MeV, and variation in the xenograft volume was recorded. mRNA and protein expression levels of Bmal1, p53, and p21 in the xenograft were measured with RT-PCR and Western blot, respectively. Results: The CNE1OE group highly expressed Bmal1 protein whereas the CNE1sh3 group was silenced by RNAi as shown with the Western blot, indicating successful transfection. The xenograft in the nude mice developed well. The CNE1OE xenograft volume was lower than that of the CNE1OENC xenograft, whereas the CNE1sh3 xenograft was larger than the CNE1shNC xenograft (P<0.05). CNE1OE, CNE1OENC, and CNE1shNC xenograft volumes shrank after being irradiated (t=4.32, 5.38, 5.16, respectively; P<0.05) and the effect was the highest in the CNE1OE group. However, there was almost no variation in xenograft volume in the CNE1sh3 group. The relative amounts of mRNA and protein of Bmal1, P53, and P21 were higher in the CNE1OE group than in the CNE1OENC group,while they were lower in the CNE1sh3 group compared to the CNE1shNC group (P<0.05). Conclusions: Overexpression of Bmal1 inhibited the growth of the CNE1 xenograft in nude mice and enhanced its radiation sensitivity whereas silencing the Bmal1 gene by RNAi promoted the growth of the xenograft and led to radiation resistance. We believe that Bmal1 overexpression leads to P53 and P21 overexpression, thereby inhibiting the growth of the xenograft.

10.
Article in Chinese | WPRIM | ID: wpr-857039

ABSTRACT

Aim To investigate the effect of deguelin on the proliferation of non-small cell lung cancer cell line A549 and nude mice. Methods CCK-8 assay was used to detect the inhibition of deguelin on proliferation of SH-SY5Y cells; Hoechst stains and Annex-inV-FITC/PI double stained method were employed to observe the apoptotic morphology and apoptotic rate; flow cytometry was applied to determine the effect of deguelin on cell cycle of A549; tumor xenograft experiment and HE staining were conducted to investigate the effect of deguelin on growth of transplanted tumor in nude mice. Results Deguelin inhibited cell proliferation of A549 dose- A nd time-dependently; Hoechst stains and AnnexinV-FITC/PI double stained further confirmed that deguelin could induce the apoptosis of A549 cells, while deguelin blocked A549 cell cycle in G2/M phase in concentration-dependent manner. The nude mice xenograft model and HE staining experiments showed that deguelin significantly inhibited the growth of xenografts in A549 nude npce (P < 0. 01). Conclusions Deguelin has a significant inhibitory effect on non-small cell lung cancer cell line A549, pointing to a basis for the study of the antitumor activity of deguelin.

11.
Acta Pharmaceutica Sinica ; (12): 2127-2133, 2020.
Article in Chinese | WPRIM | ID: wpr-825736

ABSTRACT

This study was designed to investigate the effect of dihydromyricetin (DHM) on inducing apoptosis of ovarian cancer cells A2780 through endoplasmic reticulum stress (ERS) pathway and the mechanisms involved in vitro and in vivo. A2780 cells were treated with different concentrations of DHM, and the protein expression levels of glucose-regulated protein 78 (GRP78) which is related to ERS increased, apoptotic proteins C/EBP-homologous protein (CHOP), and cysteinyl aspartate specific proteinase-12 (caspase-12) elevated. After pretreatment with ERS inhibitor, 4-phenyl butyric acid (4-PBA), following the intervention with DHM, the A2780 cell viability decreased and apoptotic rate increased. All animal welfare and experimental procedures were approved by the Animal Ethics Committee of Chongqing Medical University. Intraperitoneal injection of DHM suspension into nude mice with ovarian cancer could significantly inhibit the growth of transplanted tumor in vivo, increase the protein expression levels of GRP78, CHOP, and caspase-3. Moreover, swollen and broken endoplasmic reticulum could be observed in tumor tissues, suggesting that DHM intervention induces apoptosis mediated by ERS. The results indicated that DHM could induce apoptosis of ovarian cancer cells and inhibit the growth of transplanted tumors in nude mice, which might be related to the activation of ERS pathway.

12.
Article in Chinese | WPRIM | ID: wpr-828908

ABSTRACT

OBJECTIVE@#To investigate the difference of tumor formation in different mouse strains bearing patient-derived xenograft of esophageal squamous cell carcinoma(ESCC) and establish a better animal model for preclinical study of individualized treatment of ESCC.@*METHODS@#The tumor tissues collected from 22 ESCC patients were used to establish tumor-bearing mouse models in B-NDG (NSG) mice and BALB/c nude mice. The tumor formation rate and tumor formation time were compared between the two mouse models, and HE staining, immunohistochemistry and genome sequencing were carried out to assess the consistency between transplanted tumor tissues in the models and patient-derived tumor tissues.@*RESULTS@#The tumor-bearing models were established successfully in both NSG mice (50%, 11/22) and BALB/c nude mice (18.18%, 4/22). The average tumor formation time was significantly shorter in NSG mice than in BALB/c nude mice (75.95 91.67 days, < 0.001). In both of the mouse models, the transplanted tumors maintained morphological characteristics identical to those of patient-derived ESCC tumors. Genetic analysis showed that the xenografts in NSG mice had a greater genetic similarity to the patients' tumors than those in BALB/c nude mice ( < 0.0001).@*CONCLUSIONS@#Mouse models bearing xenografts of patient-derived ESCC can be successfully established in both NSG mice and BALB/c nude mice, but the models in the former mouse strain can be more reliable.


Subject(s)
Animals , Cell Line, Tumor , Cell Proliferation , Esophageal Neoplasms , Esophageal Squamous Cell Carcinoma , Heterografts , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Xenograft Model Antitumor Assays
13.
Braz. j. med. biol. res ; 53(11): e10067, 2020. graf
Article in English | LILACS, ColecionaSUS | ID: biblio-1132493

ABSTRACT

RU486 (mifepristone), a glucocorticoid and progesterone receptor antagonist, has been reported to exert antiproliferative effects on tumor cells. Experiments were performed to analyze the effects of RU486 on the proliferation of the human neuroblastoma, both in vitro and in vivo, using the human neuroblastoma SK-N-SH cell line. The exposure in vitro of SK-N-SH cells to RU486 revealed a dose-dependent inhibition of 3H-thymidine incorporation due to a rapid but persistent inhibition of MAPKinase activity and ERK phosphorylation. A significant decrease of SK-N-SH cell number was evident after 3, 6, and 9 days of treatment (up to 40% inhibition), without evident cell death. The inhibitory effect exerted by RU486 was not reversed by the treatment of the cells with dexamethasone or progesterone. Moreover, RU486 induced a shift in SK-N-SH cell phenotypes, with an almost complete disappearance of the neuronal-like and a prevalence of the epithelial-like cell subtypes. Finally, the treatment with RU486 of nude mice carrying a SK-N-SH cell xenograft induced a strong inhibition (up to 80%) of tumor growth. These results indicated a clear effect of RU486 on the growth of SK-N-SH neuroblastoma cells that does not seem to be mediated through the classical steroid receptors. RU486 acted mainly on the more aggressive component of the SK-N-SH cell line and its effect in vivo was achieved at a concentration already used to inhibit oocyte implantation.


Subject(s)
Humans , Animals , Rabbits , Neuroblastoma/drug therapy , Progesterone , Mifepristone/pharmacology , Glucocorticoids , Mice, Nude
14.
Article in Chinese | WPRIM | ID: wpr-841678

ABSTRACT

Objective: To establish the glioma-bearing nude mouse models, and to investigate the effect of HOXA4 on the growth of glioma U251 cells in vivo and its regulatory effect on the Wnt/β-catenin sgnal pathway and its mechanism Methods: The glioma U251 cell line stably transfected with HOXA4 siRNA (si-HOXA4) and the U251 cell line stably transfected with blank vector (si-NC) were established by lentivirus transfection The U251, si-NC, and si-HOXA4 cells were respectively inoculated under the skin of the neck and back of the BALB/c nude mice to establish the glioma-bearing nude mouse models named as control group, si-NC group, and si-HOXA4 group. The tumorigenesis of nude mice in various groups were observed and the tumor growth curve was drawn. The tumor tssue was stripped after the mice were sacrificed on the 21 th day, and the volume and wght of tumor were measured; the relative mRNA expresson amounts of HOXA4, CTNNB1, and Gsk3fS in tumor tssue of the nude mice in various groups were detected by qRT-PCR method; the expresson levels of HOXA4, β-catenin, Gsk3β, CyclinD1, and P53 proteins in tumor tissue of the nude mice in various groups were detected by immunohistochemstry (IHC) method. Results: Compared with si-NC group and control group, the volume and wght of tumor of the nude mice in si-HOXA4 group were gnificantly decreased (P<0. 05). The relative expresson amount of HOXA4 mRNA and the expresson level of HOXA4 protein in si-HOXA4 group were gnificantly lower than those in the other groups (P<0. 05). Compared with si-NC group and control group, the relative expresson amount of CTNNB1 mRNA and the expresson levels of β-catenin and CyclinDl proteins in si-HOXA4 group were significantly decreased (P<0. 05), and the expresson levels of Gsk3β and P53 proteins were gnificantly increased (P<0. 05). Conclusion: Inhibition of HOXA4 expresson in human glioma U251 cells can regulate the expressons of CyclinDl and P53 through Wnt/-catenin gnal pathway in vivo, thus inhibiting the tumor growth of glioma-bearing nude mice.

15.
Article in Chinese | WPRIM | ID: wpr-802069

ABSTRACT

Objective: To investigate the inhibitory effect of capsaicin on the growth of breast cancer MDA-MB-231 cells transplanted tumour in nude mice and its possible molecular mechanism. Method: Transplanted tumor model of breast cancer MDA-MB-231 cells in nude mice were established. Then the tumor-bearing mice were randomly divided into 4 groups:model group, and low, medium and high-dose capsaicin groups (5, 10, 20 mg·kg-1). Mice of low, medium and high-dose capsaicin groups (5, 10, 20 mg·kg-1) were intraperitoneally injected with the corresponding dose of capsaicin, and the model group was injected with the same volume of phosphate buffer saline (PBS), once every 3 days, for a total of 8 times in succession. Body weight of mice and transplantation tumor volume were measured before each injection of capsaicin. Mice of each group were put to death 24 h after the last administration, and then the tumor volume, mass and the tumor inhibitory rate were calculated. The protein expression levels of high mobility group box 1 (HMGB1) and Toll-like receptors 4(TLR4) were measured by immunohistochemistry and Western blot. Result: No significant difference was observed between each group in body weight. However, compared with the model group, capsaicin (5, 10, 20 mg·kg-1) remarkably inhibited the tumor volume and mass (PPP-1) also markedly inhibited the protein expression levels of HMGB1 and TLR4 (PConclusion: Capsaicin remarkably inhibits the growth of breast cancer MDA-MB-231 cells transplanted tumour in nude mice, and the possible mechanism may be related to the down-regulation of HMGB1 and TLR4 at the protein level.

16.
Article in Chinese | WPRIM | ID: wpr-801997

ABSTRACT

Objective: To observe the inhibitory effect of astragalus polysaccharides (APS) on growth of human breast cancer MDA-MB-231 xenograft tumor in nude mice and its effect on the apoptosis of tumor cells, in order to study the effect of APS on growth and induction of apoptosis of triple negative breast cancer MDA-MB-231 and its possible molecular mechanism. Method: Human breast cancer cell MDA-MB-231 was inoculated into the right axillary subcutaneous of BALB/c-nu female nude mice to establish the transplanted tumor model of breast cancer. Eighteen nude mice were randomly divided into 3 groups:model group (saline per day), low-dose APS group (200 mg·kg-1 APS per day), and high-dose APS group (400 mg·kg-1 APS per day), with 6 rats in each group. The drug was administered by gavage (200 μL) daily for 21 days. In the experiment, the length and diameter of breast cancer transplanted tumor were measured every two days, and the tumor volume was recorded and calculated. At the end of the experiment, the changes of tumor mass and tumor volume of the low and high-dose APS groups and the model group were observed and compared, and the tumor inhibition rate was calculated. The cell morphology in tumor tissue was observed by hematoxylin-eosin (HE) staining, and Terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) was used to verify the apoptosis of breast cancer tissues. The expressions of apoptosis-related proteins, such as B-cell lymphoma/leukemia-2 protein (Bcl-2), Bcl-2 associated X protein (Bax), Caspase in tumor tissues was detected by Western blot. Result: The tumor volume of breast cancer decreased in the low and high-dose APS groups, and the tumor inhibition rates were 37.9%and 57.57%, respectively, with statistically significant differences from the model group (PP0.01). HE of tumor tissue cells showed that APS led to obvious morphological changes, with apoptosis in the tissue cells. TUNEL staining showed that the apoptosis rate of tumor cells in APS intervention groups was higher than that in control group. Western blot showed that expression of Bcl-2 protein decreased(PPPPConclusion: APS can effectively inhibit the growth of MDA-MB-231 breast cancer xenografts in nude mice and induce apoptosis in human breast cancer MDA-MB-231 cells. The mechanism may be related to the effect of APS on expressions of apoptosis-related proteins Bcl-2, Bax, Caspase-9 and Caspase-7 in breast cancer cells.

17.
Cancer Research and Clinic ; (6): 799-804, 2019.
Article in Chinese | WPRIM | ID: wpr-800716

ABSTRACT

Objective@#To investigate the effect of cinobufacin combined with As2O3 on the angiogenesis of subcutaneous transplantation tumor in colorectal carcinoma BALB/C nude mice.@*Methods@#The colorectal carcinoma transplantation tumor model of BALB/C nude mice was established and divided into 4 groups, 5 mice in each group. The growth state of nude mice was observed by injecting the corresponding reagents into the tumor in As2O3 group, cinobufacin group, cinobufacin combined As2O3 group and the blank control group (replaced by phosphate buffer), respectively. The nude mice were killed two weeks later, and the tumor tissues, liver and kidney tissues and orbital vein blood were taken. The tumor volume inhibition rate and mass inhibition rate of nude mice were calculated. The histomorphology of tumor, liver and kidney and blood routine were detected. The expressions of vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), fibroblast growth factor-basic (b-FGF) and CD105 in transplanted tumor tissues were detected by using immunohistochemistry method, and the microvascular density (MVD) of transplanted tumor in nude mice was evaluated. Western blot method was used to detect the protein expression levels of VEGF, EGFR and b-FGF.@*Results@#After 2 weeks of administration, the tumor volume and tumor mass in As2O3 group, cinobufacin group and cinobufacin combined As2O3 group were lower than those in the blank control group. The volume inhibition rate was 16%, 17%, 72%, and the mass inhibition rate was 31%, 33%, 78%, respectively, and the difference was statistically significant (all P < 0.01); there was no statistical difference between As2O3 group and cinobufacin group in the tumor volume and tumor mass (P > 0.05), and cinobufacin combined As2O3 group had the most obvious therapeutic effects (all P < 0.05). The immunohistochemistry method showed that the expressions of VEGF, EGFR, b-FGF and MVD were decreased in As2O3 group, cinobufacin group and cinobufacin combined As2O3 group compared with the blank control group, and there were statistical differences of the four groups (all P < 0.05). There was no statistical difference of the marker changes in As2O3 group and cinobufacin group (all P > 0.05), and cinobufacin combined As2O3 group had the most significant decrease in the marker changes, and there was a significant difference compared with the other groups (all P < 0.05). Western blot showed that compared with the blank control group, the other three groups could downregulate the protein expressions of VEGF, EGFR and b-FGF in the transplanted tumor of nude mice. And the decreasing expression of each protein in cinobufacin combined As2O3 group was the most significant. Pathomorphology examination showed that the histomorphology of liver and kidney of the four groups of nude mice was normal. Blood routine examination showed that compared with the blank control group, the white blood cell count of nude mice in other groups was decreased, and the difference was statistically significant (all P < 0.05). The white blood cell count of cinobufacin combined As2O3 group was decreased most significantly; there was no statistically significant difference in hemoglobin and platelet count among the four groups (all P > 0.05).@*Conclusions@#Cinobufacin and As2O3 show synergistic effects in the tumor angiogenesis and inhibition of transplantation tumor growth of colorectal cancer BALB/C nude mice. Moreover, cinobufacin and As2O3 have no obvious toxicity to the hepatic, kidney and hematopoietic tissues.

18.
Chinese Pharmaceutical Journal ; (24): 373-381, 2019.
Article in Chinese | WPRIM | ID: wpr-858054

ABSTRACT

OBJECTIVE: To investigate the inhibitory effect of compound Daqiqi decoction(CDQD) combined with cisplatin on subcutaneously transplanted ovarian cancer in nude mice and its related mechanisms. METHODS: The 60 subcutaneously transplanted model of nude mice was established with human ovarian cancer cell line SKOV3, and divided into 6 groups randomly, each group of 10 nude mice, which were model group that was treated with saline, CDQD low dose group with the CDQD dosage of 15.16 g•kg-1, CDQD medium dose group with the CDQD dosage of 30.33 g•kg-1, CDQD high dose group with the CDQD dosage of 60.66 g•kg-1, cisplatin group with the DDP dosage of 3 mg•kg-1 and combined group that was treated with the CDQD dosage of 30.33 g•kg-1 and the DDP dosage of 3 mg•kg-1. Cisplatin was administered once every 3 d, and the remaining drugs were administered once a day. Then,the tumor-bearing mouse model was given the corresponding drugs for 14 consecutive days, and the tumor volume was measured every 3 d. After the end of treatment, the tumor was removed and weighed. The morphology of the tumor cells were observed by HE staining. The apoptosis of tumor cells was detected by TUNEL method. The mRNA and protein expression of miR939 and STAT3 and VEGFA and EGFR in tumor tissues were detected by real-time fluorescence quantitative PCR and Western-blot. RESULTS: The tumor volume and the tumor weight of the treated groups were all decreased(P<0.01). Compared with the model group, the tumor volume and tumor weight of the combined group were less than those of the other groups(P<0.01), and the apoptosis rate of the combined group was significantly higher than other groups(P<0.01).The expression of miR939 and STAT3 and VEGFA were down-regulated and the expression of EGFR were up-regulated in the treatment groups. Compared with the model group, the expression of MiR-939, STAT3 and VEGFA were down-regulated and the expression of EGFR was increased in the treatment group. MiR-939, STAT3, VEGFA expression in the combined group was the lowest(P<0.05), and the EGFR expression was highest(P<0.05). CONCLUSION: Studies have shown that CDQD can inhibit ovarian cancer subcutaneously transplanted tumor in nude mice, and its mechanism may be related to inhibition of MiR-939/STAT3 pathway activation, down-regulation of VEGFA, and up-regulation of EGFR expression. The inhibitory effect of CDQD on ovarian cancer tissues has a concentration dependence. And the combination of CDQD and DDP can enhance the anti-tumor effect of DDP and reduce the side effects of DDP on tumor-bearing mice.

19.
China Pharmacy ; (12): 601-607, 2019.
Article in Chinese | WPRIM | ID: wpr-817059

ABSTRACT

OBJECTIVE: To study the anti-tumor effect of anemoside B4 (AB4) on hepatocellular carcinoma Huh-7 and tumor-bearing nude mice and its mechanism. METHODS: Huh-7 cells were divided into AB4 treatment group, negative control group (constant volume of culture medium) and positive control group (5-fluorouracil 50 μmol/L). The inhibitory rate of Huh-7 cell proliferation was tested by MTT after treated with 0, 3, 6, 12, 25, 50, 100, 200, 400, 800, 1 600 μmol/L AB4 for 12, 24, 36, 48 h; IC50 were calculated. The number of clone cells was evaluated by clone formation tests after Huh-7 cells were treated with 50 μmol/L AB4 for 24 h. The apoptosis rate of Huh-7 cells was tested by Annexin Ⅴ-FITC/PI double staining after treated with 50 μmol/L AB4 for 24 h. The expression of apoptotic proteins such as Bcl-2, Bax, Caspase-3, Cleaved Caspase-3 and Cleaved PARP were tested by Western blot after Huh-7 cells were treated with 50 μmol/L AB4 for 24 h. Tumor-bearing nude mice were randomly divided into negative control group (normal saline), positive control group (5-fluorouracil 50 mg/kg), AB4 lwo-dose, medium-dose and high-dose groups (25, 50, 100 mg/kg), with 3 mice in each group. They were given relevant medicine intraperitoneally, once a day, for consecutive 19 d. The growth of tumor was observed, and anti-tumor rate was also calculated. HE staining was used to observe the morphology of tumor cells. RESULTS: The inhibition rate of AB4 on Huh-7 cell proliferation increased with the increase of concentration of AB4, but it did not increase significantly after reaching 50 μmol/L; it increased with the increase of time, but it did not increase significantly after 24 h. The IC50 of AB4 was (56.76±1.756) μmol/L. Compared with negative control group, the number of clone cells was decreased significantly after Huh-7 cells were treated with 50 μmol/L AB4 for 24 h, while the protein expression of Bcl-2 was decreased significantly (P<0.05); the apoptotic rate, the protein expression of Bax, Caspase-3, Cleaved Caspase-3 and Cleaved PARP were increased significantly (P<0.05 or P<0.01), there was no statistical significance, compared with positive control group. Compared with negative control group, the volume of tumor was decreased significantly in AB4 low-dose, medium-dose and high-dose groups, positive control group (P<0.05); the outline of tumor cells become more and more blurred; there were nuclear pyknosis, unclear nucleoplasm and nuclear fragmentation; its anti-tumor rates were 25.93%, 39.15%, 46.26%, 42.83%, respectively. CONCLUSIONS: AB4 inhibits Huh-7 cells and tumor-bearing mice, the mechanism of which may be associated with up-regulating the proportion of Bax/Bcl-2, activating Caspase-3, cracking PARP and inducing apoptosis.

20.
Article in Chinese | WPRIM | ID: wpr-756210

ABSTRACT

Objective To compare the changes in IFN-γ, IL-4, IL-10 and IL-1β expression at mRNA level in gastric mucosae of BALB/c, C57BL/6 and nude mice at different stages of Helicobacter heilmannii ( H. heilmannii) infection, and to investigate the types of induced immune responses. Methods Each kind of mice was randomly divided into two groups: infection ( n=30 ) and control ( n=6 ) groups. Those in the infection groups were intragastrically inoculated with H. heilmannii strains to establish long-term stable mouse infection models. Gastric mucosa tissues were collected at weeks 4, 8, 12, 24 and 36 and ana-lyzed by semi-quantitative RT-PCT to detect the expression of IFN-γ, IL-4, IL-10 and IL-1βat mRNA lev-el. Results In the early stage of infection (weeks 4-12), INF-γ, IL-4, IL-10, and IL-1β expression at mRNA level in the gastric mucosae of BALB/c and C57BL/6 mice were significantly increased compared with those of the control groups (P<0. 05). IL-4, IL-10 and IFN-γexpression peaked at weeks 8-12, while IL-1βexpression reached the peak at week 4. After 12 weeks, IFN-γexpression at mRNA level in BALB/c mice was significantly decreased, but showed no significant change in C57BL/6 mice. IL-4 expression at mRNA level in C57BL/6 mice at the late stage of infection (week 36) was lower than that in the correspond-ing control group (P<0. 05). Expression of IFN-γ, IL-4, IL-10 and IL-1β at mRNA level in nude mice were all higher than those in the control group, and there were significant differences in IL-1βand IL-4 ex-pression between groups (P<0. 05). Conclusions Expression of cytokines in H. heilmannii-infected mice increased over time. IFN-γ-mediated Th1 immune responses were the predominant immunity induced by H. heilmannii infection. Immune responses to H. heilmannii infection varied with the kinds of mice. C57BL/6 mice showed mainly Th1 cell immune responses, while Th1/Th2 mixed immune responses were induced in BALB/c mice.

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