Cervical Cancer In Nude Mice Research Articles

DYNLT3 overexpression induces apoptosis and inhibits cell growth and migration via inhibition of the Wnt pathway and EMT in cervical cancer.

The role of the dynein light chain Tctex-type 3 (DYNLT3) protein in the biological behavior of cervical cancer and its relative molecular mechanisms were investigated. Immunohistochemical staining was used to detect DYNLT3 protein expression in cervical cancer tissues. Cell proliferation and apoptosis rates and invasiveness and migratory capacities were determined by CCK-8 assays, BrdU staining assays and colony formation assays, fluorescence activated cell sorting (FACS), wound healing assays, and Transwell invasion assays of cervical cancer cells after DYNLT3 modulation. The expression levels of Wnt signaling pathway- and EMT-related proteins were examined by Western blotting. Furthermore, the effects of DYNLT3 on the tumorigenicity and metastasis of cervical cancer in nude mice were analyzed by performing immunohistochemistry, and we found that the expression level of the DYNLT3 protein was higher in human normal cervical tissues than in cervical cancer tissues. Overexpression of DYNLT3 obviously attenuated the proliferation, migration and invasion of CaSki and SiHa cells, and promoted cell apoptosis. Upregulation of DYNLT3 expression markedly decreased the expression of Wnt signaling pathway-related proteins (Dvl2, Dvl3, p-LRP6, Wnt3a, Wnt5a/b, Naked1, Naked2, β-catenin and C-Myc) and EMT-related proteins (N-cadherin, SOX2, OCT4, vimentin and Snail), and increased the expression of E-cadherin and Axin1. However, the opposite results were observed after down-regulation of DYNLT3 expression. Up-regulation of DYNLT3 expression significantly inhibited tumor growth in a nude mouse model, while downregulation of DYNLT3 showed the opposite results. In addition, the major metastatic site of cervical cancer cells in mice was the lung, and downregulation of DYNLT3 expression increased cancer metastasis in vivo. DYNLT3 exerted inhibitory effects on cervical cancer by inhibiting cell proliferation, migration and invasion, promoting cell apoptosis in vitro, and inhibiting tumor growth and metastasis in vivo, possibly by suppressing the Wnt signaling pathway and the EMT.

Long non-coding RNA HAND2-AS1 delays cervical cancer progression via its regulation on the microRNA-21-5p/TIMP3/VEGFA axis.

Cervical cancer is a common cause of cancer-related mortality in women. Mounting evidence suggests that long non-coding RNAs (lncRNAs) function vitally in many cancers. In this study, we discovered that the regulation of the heart and neural crest derivatives expressed 2-antisense RNA 1 (HAND2-AS1) in cervical cancer. RT-qPCR was conducted to detect the expression of HAND2-AS1 and microRNA-21-5p (miR-21-5p). The relationship of HAND2-AS1 and miR-21-5p was identified by dual-luciferase reporter gene assay. The roles of HAND2-AS1, miR-21-5p and tissue inhibitor of metalloproteinases-3 (TIMP3) in cervical cancer were accessed via gain- and loss-of-function approaches. The expression of related proteins in the vascular endothelial growth factor A (VEGFA) signaling pathway was detected through Western blot analysis. Finally, xenografts of cervical cancer in nude mice were established to assess the effect of HAND2-AS1 on tumorigenesis in vivo. HAND2-AS1 and TIMP3 were downregulated in cervical cancer, which were identified to be associated with a poor prognosis of patients with cervical cancer. Moreover, HAND2-AS1 was upregulated the expression of TIMP3 through competitively binding to miR-21-5p. Overexpressed HAND2-AS1 or downregulated miR-21-5p inhibited cell proliferation, migration, and invasion while promoting cell apoptosis, in association with increased expression of proteins in VEGFA signaling pathway. These changes were reversed by silencing of TIMP3. Overexpressed HAND2-AS1 reduced the tumor formation ability in nude mice. In summary, HAND2-AS1 may exert inhibitory effects on cervical cancer cell growth and cervical cancer development through its regulation on the miR-21-5p/TIMP3/VEGFA axis. This highlights that HAND2-AS1 may serve as a potential target for cervical cancer diagnosis and treatment.

LncRNA HAND2-AS1 represses cervical cancer progression by interaction with transcription factor E2F4 at the promoter of C16orf74.

Cervical cancer is one of the major malignancies, the pathophysiology and progression of which remain to be scarcely understood. Long non‐coding RNAs (lncRNAs) have been previously implicated in the progression of cervical cancer. Here, the purpose of this study was to identify whether lncRNA heart‐ and neural crest derivative‐expressed 2‐antisense RNA 1 (HAND2‐AS1) affect the development of cervical cancer through regulation of chromosome 16 open reading frame 74 (C16orf74) by mediating a transcription factor E2F4. RT‐qPCR was performed to determine the expression of HAND2‐AS1 in cervical cancer cells. Then, cervical cancer cells were treated with HAND2‐AS1 or si‐E2F4 RNA, or C16orf74, after which the proliferation, colony formation, migration and invasion were detected. Moreover, the binding between HAND2‐AS1 and E2F4 or between E2F4 and C16orf74 was explored. Finally, the tumorigenesis of cervical cancer cells was measured in nude mice with altered HAND2‐AS1/E2F4/C16orf74 expression. HAND2‐AS1 exhibited poor expression in cervical cancer, and HAND2‐AS1 overexpression suppressed the proliferation, colony formation, migration and invasion of cervical cancer cells. In addition, HAND2‐AS1 was found to recruit transcription factor E2F4 to C16orf74 promoter region and down‐regulate C16orf74 expression. Lastly, HAND2‐AS1/E2F4/C16orf74 modulated the tumorigenesis of cervical cancer in nude mice. In conclusion, this study provided evidence on the inhibitory effect of HAND2‐AS1 on the development of cervical cancer through the suppression of C16orf74 expression by recruiting transcription factor E2F4. This study highlights the potential of lncRNA HAND2‐AS1 as a target in the treatment of cervical cancer.

Bcl-2 associated athanogene 3 affects the epithelial-mesenchymal transition in human cervical cancer

Objective: To investigate the expression of Bcl-2 associated athanogene 3 (BAG3) in cervical cancer tissues and cells and its role in epithelial mesenchymal transition (EMT) of cervical cancer. Methods: (1) Cervical cancer samples were collected from September 2015 to March 2017 in the Qilu Hospital of Shandong University and Shangdong Provincial Hospital. While, 50 normal tissues were collected from August 2015 to March 2017 in the Dezhou Municiple Hospital, which were obtained from patients with uterine myoma underwent hysterectomy and patients with cervical biopsy. Reverse transcription (RT)-PCR and western blot were used to detect the expression of BAG3 mRNA and protein, and their clinical significances were analyzed. (2) The expression of BAG3 mRNA and protein was detected using RT-PCR and western blot method in HeLa and SiHa cell lines and normal cervical epithelial cells. The experiment was divided into two groups, BAG3 small interfering RNA transfected group (si-BAG3) and the control group transfected with small interfering RNA (siRNA). Cell counting kit 8 (CCK-8) analysis was used to detect cell proliferation of two groups. Wound-healing and transwell assay were used to detect the migration and invasion ability of HeLa and SiHa cells. The xenograft model of cervical cancer in nude mice was used to observe the effect of BAG3 on tumor xenografts and the tumor-related biomarkers were tested by western blot. Results: (1) The expression levels of BAG3 mRNA and protein in cervical carcinoma tissues were 1.20±0.15 and 1.10±0.16, which were significantly higher than that in normal cervical tissue, 0.23±0.04 and 0.29±0.03 (both P<0.01). The results showed that the expression levels of BAG3 mRNA and protein were significantly correlated with cervical carcinoma staging and lymph node metastasis (P<0.05).However, its expression was not correlated with the patient's age, pathological grade, and diameter of tumor (all P>0.05). (2) Compared with normal cervical epithelial cells, the expression of BAG3 mRNA and protein levels in HeLa and SiHa cells were significantly increased (P<0.01), the expression levels of BAG3 mRNA and protein in HeLa and SiHa cells transfected with si-BAG3 were significantly lower than that in control group (all P<0.01). After post-transfected 72 hours, A value of HeLa and SiHa with transfection were significantly lower than those in control group [(0.88±0.08) vs (1.22±0.13), (0.92±0.09) vs (1.35±0.12); both P<0.01]. After post-transfected 24 hours, the migration level of HeLa and SiHa cells with transfection were significantly lower than those in the control group [(20.1±2.1)% vs (58.6±5.6)%, and (21.1±2.1)% vs (61.7±5.4)%; both P<0.01]. The transmembrane cell number in HeLa and SiHa cells with transfection were 76±11 and 71±8, which were significantly less than those in control group (131±12 and 129±14; both P<0.01). After the inoculation into nude mice, tumor formation time of HeLa and SiHa cells with transfection were (9.5±0.5) and (10.5±1.3) days, respectively, which were significantly longer than those in control group [(4.5±0.5) and (5.2±1.1) days; both P<0.05]. Compared with those in the control group, the expression level of Slug, N-cadherin and matrix metalloproteinase-2 (MMP-2) protein in HeLa and SiHa cells with transfected in tumor tissues were significantly decreased (all P<0.01), while the expression level of E-cadherin protein was significantly increased (P<0.01). Conclusion: BAG3 could be involved in the proliferation, migration and invasion of cervical cancer cells by affecting cervical cancer EMT, and BAG3 may be an effective target for the treatment of cervical cancer.

Experimental study on anti-tumor effect of high intensity focused ultrasound ablation combined with liquid fluorocarbon nanoparticle on cervical cancer

Objective To investigate the therapeutic effect of high intensity focused ultrasound(HIFU) ablation combined with homemade liquid fluorocarbon nanoparticles on cervical cancer in nude mice. Methods The cell experiment was divided into three groups: a, control group; b, HIFU group; c, HIFU+ PFB nanoparticle group, and the viability of cells was detected using CCK-8 reagent. The mice were also divided into three groups: A, 0.9%NaCI group; B, HIFU+ 0.9%NaCI group; C, HIFU+ PFB nanoparticle group. The tumors were removed and underwent triphenyl tetrazolium chloride(TTC) staining, and the necrosis area was measured. Histopathological changes of the tumors were examined by light microscopy. Results After HIFU irradiation, the viability rate of group c was (40.5±9.7)%, it was lower than that of group b (77.7±8.5)% (P<0.05) and that of group a(100±4.8)% (P<0.05). TTC staining of tumor showed a large scale of necrotic tissue in group C. The necrosis ratio of the three groups was 0%, (34.14±12.2)% and (65.97±25.1)%, respectively (P<0.05). HE staining showed karyorrhexis or an absence of nuclei in group B and group C, which demonstrated the coagulation necrosis. Conclusions HIFU ablation combined with liquid fluorocarbon nanoparticles can effectively treat the xenograft model of the human cervical carcinoma in nude mice. Key words: High intensity focused ultrasound; Nanoparticles; Uterine cervical neoplasms; Liquid fluorocarbon

EIF3f reduces tumor growth by directly interrupting clusterin with anti-apoptotic property in cancer cells.

Clusterin is a secretory heterodimeric glycoprotein and the overexpression of secretory clusterin (sCLU) promotes cancer cell proliferation and reduces chemosensitivity. Therefore, sCLU might be an effective target for anticancer therapy. In the current study, we identified eIF3f as a novel CLU-interacting protein and demonstrated its novel function as a CLU inhibitor. The overexpression of eIF3f retarded cancer cell growth significantly and induced apoptosis. In addition, eIF3f interacted with the α-chain (1–227) of sCLU. This interaction blocked modification of psCLU, thereby decreasing the expression and secretion of α/β CLU. Consequently, the overexpression of eIF3f suppressed Akt and ERK signaling and subsequently depleted CLU expression. In addition, eIF3F stabilized p53, which increased the expression of p21 and Bax. Interestingly, the expression of Bax was increased without the activation of p53. eIF3f injected into a xenograft model of human cervical cancer in nude mice markedly inhibited tumor growth. The identification of this novel function of eIF3f as a sCLU inhibitor might open novel avenues for developing improved strategies for CLU-targeted anti-cancer therapies.

Ameliorative effect of ginsenoside RT-5 on CDDP-induced nephrotoxicity

The aim of this study is to explore whether RT-5, one novel active ginsenosides from Ginseng, has protective effects against cisplatin (CDDP)-induced nephrotoxicity in vivo. One model of acute renal failure induced by CDDP in rats and one model of xenograft tumors of human cervical cancer in nude mice are established. Four groups are assigned: control, CDDP, RT-5, CDDP plus RT-5, in which the RT-5 is administered via oral gavage 24 h before CDDP intraperitoneal injection. The significant increase in blood urea nitrogen and creatinine are induced by CDDP treatment at 6 mg/kg, which are attenuated by pre-treated with RT-5 at 10 mg/kg. RT-5 could ameliorate CDDP-induced morphological damages in kidney by PAS staining, and reduce tubular apoptosis evaluated by TUNEL staining. Pretreatment with RT-5 notably inhibits CDDP-induced oxidative stress in kidney tissues. Interestingly, RT-5 does not interfere with the in vivo anti-cancer effects of CDDP against the growth of xenograft tumors in nude mice. These data suggest that co-treatment RT-5 with CDDP might attenuate the following nephrotoxicity without inhibiting its anti-tumor efficiency, which could provide one novel strategy for cancer treatment in clinics.

Abstract 1229: The advantages of patient-derived orthotopic xenograft (PDOX) of metastatic cervical cancer for individualized therapy compared to the PDX model

Abstract In this study, we demonstrated the advantage of a patient derived orthotopic xenograft (PDOX) compared to a subcutaneous patient cervical cancer. We originally established the PDX (F1) from the patient with a metastatic HER-2-positive cervical cancer in nude mice. We implanted to the small fragments of F1 PDX to 30 nude mice subcutaneously and 30 nude mice orthotopically to examine the efficacy of various drugs for this tumor. Six weeks after implantation, the mice in each model were randomized to 5 groups and treated in the following groups: (1) Trastuzumab (20 mg/kg, ip, weekly, 5 weeks) + Lapatinib (100 mg/kg, orally, daily, 5 weeks); (2) Entinostat (5 mg/kg, orally, daily, 5 weeks); (3) Trastuzumab (20 mg/kg, ip, weekly, 5 weeks) + Lapatinib (100 mg/kg, orally, daily, 5 weeks) + Entinostat (5 mg/kg, orally, daily, 5 weeks); (4) Carboplatin (30 mg/kg, ip, weekly, 5 weeks); and (5) saline (vehicle/control, ip, weekly, 5 weeks). The relative tumor volume, compared to time zero, of each group in the PDX model was as follows: (1) 1.23 ± 0.50; (2) 3.07 ± 1.79; (3) 0.38 ± 0.18; (4) 0.39 ± 0.23; (5) 10.23 ± 2.91. All regimens had significant efficacy compared to the untreated control group. The Trastuzumab/Lapatinib/Entinostat combination and Carboplatin were the most effective regimens in the subcutaneous model. The relative primary tumor volume of each group in the PDOX model was as follows: (1) 1.00 ± 0.23; (2) 3.25 ± 1.16; (3) 0.54 ± 0.34; (4) 0.48 ± 0.39; (5) 4.45 ± 1.09. The metastatic tumor weight of each group in PDOX model was as follows: (1) 5.7 ± 9.8 mg; (2) 20.1 ± 25.1 mg; (3) 4.7 ± 8.1 mg; (4) 0 ± 0 mg; (5) 112.0 ± 32.5 mg. All regimens had significant efficacy on the primary tumors compared to the control group except for the Entinostat alone group. However, Entinostat alone significantly reduced the metastatic tumor weight compared to the control. The Trastuzumab/Lapatinib/Entinostat combination and Carboplatin were the most effective regimens on both primary and metastatic tumors in the PDOX model. However, only the PDOX model metastasized and could be used to discover the anti-metastatic activity of the Entinostat alone. These results suggest that PDOX is the suitable model for individualized therapy as well as drug discovery for metastatic cervical cancer. Citation Format: Yukihiko Hiroshima, Yong Zhang, Ali Maawy, Sho Sato, Takashi Murakami, Mako Yamamoto, Fuminari Uehara, Shinji Miwa, Shuya Yano, Masashi Momiyama, Takashi Chishima, Kuniya Tanaka, Michael Bouvet, Itaru Endo, Robert M. Hoffman. The advantages of patient-derived orthotopic xenograft (PDOX) of metastatic cervical cancer for individualized therapy compared to the PDX model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1229. doi:10.1158/1538-7445.AM2014-1229

Mechanism of anti-tumor effect of HIF-1α silencing on cervical cancer in nude mice

Objective The aim of our study was to observe the anti-tumor effect of silencing the expression of HIF-1α on cervical cancer in nude mice and to explore its mechanism of action.

Mechanism of anti-tumor effect of HIF-1alpha silencing on cervical cancer in nude mice

To observe the anti-tumor effect of silencing the expression of HIF-1alpha on cervical cancer in nude mice and to explore its mechanism of action. Human cervical cancer cell line Siha cells were divided into 3 groups: mock control group, control group transfected with scrambled sequence plasmid, and experimental group transfected with pU-HIF-1alpha-shRNA eukaryotic expression plasmid. Cultured cells of the three groups were inoculated in nude mice to establish cervical cancer-bearing nude mice. HIF-1alpha RNAi assay was performed to evaluate the tumor-suppressive effect of HIF-1alpha silencing on cervical cancer-bearing nude mice. Immunohistochemistry and Western blot were used to observe the distribution and protein expression of HIF-1alpha and GLUT1, while RT-PCR was adopted to detect the gene expression of HIF-1alpha, GLUT1 and HKII. The product of glycolysis (lactic acid) and apoptosis in tumor cells were detected by colorimetry and semi-quantitative TUNEL staining, respectively. The tumor growth in experimental group was significantly slower than that in the two control groups (P < 0.05). On the 50th day after transplantation, the tumor weight in the experimental group was (1.90 +/- 0.28) g, significantly lower than (2.95 +/- 0.77) g in the control group and (2.54 +/- 0.56) g in the mock group (P < 0.01). In the experimental group, the gene and protein levels of HIF-1alpha were 0.45 +/- 0.04 and 1.25 +/- 0.92, and the levels of GLUT1 were 0.32 +/- 0.02 and 1.25 +/- 0.48, respectively. Both indicators in HIF-1alpha and GLUT1 were lower than that in the two control groups (P < 0.05). The expression levels of HKII gene and lactic acid in the experimental group were lower than that in the two control groups (P < 0.05), but the apoptotic cells were much more numerous in the experimental group than that in matched control groups (P < 0.01). The gene therapy by siRNA targeted silencing of HIF-1alpha may down-regulate its downstream genes GLUT1 and HKII expression, therefore, to reduce the tumor glycolysis activity and promote tumor cell apoptosis, and exert a tumor-suppressing effect in vivo.

Inhibition of HPV 16 E6 oncogene expression by RNA interference in vitro and in vivo

Substantial studies have demonstrated that the initiation and progression of cervical cancer were closely associated with human papillomavirus (HPV) E6 and E7 oncogenes. The therapeutic strategy with ribozyme or antisense oligonucleotides to inhibit the expression of HPV E6 or E7 oncogenes showed effect to some degree, but problems such as low efficiency, short-period maintenance, and high cost still remain. The aim of this study was to investigate in vitro and in vivo the effect of HPV 16 E6 small interfering RNA (HPV 16 E6 siRNA) on cervical cancer cell line CaSki cells. The specific siRNA of HPV 16 E6 was synthesized and transfected into CaSki cells by liposome. The number of apoptotic cells, HPV 16 E6 messenger RNA (mRNA) level, and E6 protein expression were measured before and after the transfection by flow cytometry, reverse transcriptase-polymerase chain reaction, and Western blot, respectively. Cervical cancer in nude mice was established, and siRNA was injected directly into the nude mice peritoneal cavity or subcutaneous tumor. The efficiency of siRNA was evaluated by tumor volume change, HPV 16 E6 protein expression, and apoptosis of tumor cells. Apoptosis rate of CaSki cells at days 1, 2, 5, and 9 after siRNA transfection were 7.7%, 11.8%, 37.4%, and 12.6%, respectively. The mRNA level of HPV 16 E6 at the same time points were reduced by 77%, 83%, 59%, and 41%, respectively. But the mRNA level of beta-actin, as an internal control, showed no significant change. The inhibition rates of E6 protein synthesis at days 1, 2, 5, and 9 after the transfection were 79.7%, 80.4%, 71.3%, and 57.4%, respectively, whereas the protein levels of Lamin A/C, as internal control, had no change. In vivo, E6 siRNA administration groups showed a dramatic effect in inhibiting tumor growth, suppressing expression of E6 protein, and inducing tumor necrosis and apoptosis as compared with the control group. Direct injection of siRNA into subcutaneous tumor resulted in tumor suppression effect similar to that via the peritoneal cavity, and with additional injection better results could be achieved in cervical cancer CaSki cells. RNA interference exists, and the interference to HPV 16 E6 is specific and highly efficient both in vitro and in vivo.