Cervical Cancer Xenograft Model Research Articles

Abstract B79: Evaluation of active hexose correlated compound (AHCC) for the prevention or delay of tumor growth in human cervical cancer xenograft model

Abstract Purpose: Active hexose correlated compound (AHCC) is a mixture of polysaccharides, amino acids, lipids and minerals extracted from the culture of the basidiomycete mushroom Lentinula edodes (shiitake) that has been proposed to have many health benefits including both immunomodulatory and anti-tumor effects. In clinical studies AHCC has demonstrated numerous immunomodulating and potential restorative effects on natural killer (NK) cells, macrophages and cytokines. The objectives of this study were to evaluate if daily treatment with AHCC would eradicate human papillomavirus (HPV) 16/18 expression and prevent or delay cervical tumor growth using human xenograft mouse model. Methods: Selected cervical cancer cells, SiHa (HPV 16/18 positive), and C-33A (HPV negative) were treated in vitro with a single dose AHCC 0.42 mg/mL and incubated for 72 hours. In the second study AHCC dose was repeated once every 24 hours for total of seven days. This was followed by a three arm in vivo study in two xenograft cervical cancer mouse models, SiHa (HPV 16/18 positive), and C-33A (HPV negative), in which each cell line had ten mice for the treatment arm, vehicle control arm and no treatment arm. Mice in the treatment arm received 50 mg/kg AHCC in 0.25 mL of sterile water every day for seven days before the injection of the tumor cells and until the completion of the study. Tumors were measured three times per week. After 90 days of treatment, there was a 30 day observation period to evaluate the potential for recurrence of the HPV infection and the impact on tumor growth. At the end of the study, tumors were extracted and RT-PCR was completed on DNA samples from extracted protein to evaluate the HPV expression. Results: In vitro treatment with a single dose of AHCC for 72 hour incubation suppressed HPV expression in the first 24 hours but then HPV expression recovered by 48 hours. However, with continuous in vitro exposure, sustained HPV suppression was observed. In the in vivo animal studies, expression of HPV was eradicated with once daily AHCC dosing for 90 days and no detection of HPV expression was sustained after 30 days off treatment. In addition, AHCC daily treatment was associated with a 15.9% decrease in SiHa (HPV 16/18 positive) tumor growth compared to the untreated control (P< 0.05). AHCC did impact the growth rate of the C-33A (HPV negative) tumors. Conclusion: In conclusion, these data suggest daily dosing of AHCC will eradicate HPV 16/18 infections and may have a role in the prevention of HPV-related cervical cancer. Furthermore, there is a potential for the addition of AHCC to primary treatment regimens for cervical cancer, which may potentially improve response rates and prevent recurrence. A confirmatory pilot study in HPV positive women is underway. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B79.

The synergistic therapeutic effect of cisplatin with Human papillomavirus E6/E7 short interfering RNA on cervical cancer cell lines in vitro and in vivo

Human papillomavirus (HPV) types 16 and 18 are the major etiologic factors in the development of cervical epithelial neoplasia. Our study was designed to validate antiviral short interfering RNA (siRNA) targeting the E6 and E7 oncogenes as a potential chemosensitizer of cisplatin (cis-diaminedichloroplatinum II; CDDP) in cervical carcinoma. Specifically, the therapeutic efficacy of combination of CDDP and E6/E7-specific siRNA was assessed in an in vivo cervical cancer xenograft models. The combination of CDDP and E6/E7-specific siRNA had greater efficacy than the combination of CDDP and E6-specific siRNA especially in terms of inducing cellular senescence. Through in vitro and in vivo experiments, the mechanism of synergy between these two treatments was revealed, demonstrating that the combination of E6/E7-specific siRNA and CDDP therapy was significantly superior to either modality alone. In vitro, long-term exposure of HeLa cells to the combination of CDDP and E6/E7-specific siRNA induced apoptosis and cellular senescence. In vivo, E6/E7-specific siRNA potentiated the antitumor efficacy of CDDP via induction of apoptosis, senescence and antiangiogenesis. Our results suggest that E6/E7-specific siRNA may be an effective sensitizer of CDDP chemotherapy in cervical cancer.

Orthotopic Xenograft Model of Cervical Cancer for Studying Microenvironmental Effects on Metastasis Formation and Response to Drug Treatment

Cancers arising in the uterine cervix are usually squamous cell carcinomas that develop from preneoplastic lesions. They invade locally, and then typically metastasize to the regional lymph nodes and eventually to distant sites. Orthotopically grown xenografts are technically challenging to perform, but recapitulate the clinical situation to a greater extent than xenografts grown at subcutaneous or intramuscular sites. Thus, orthotopic xenografts develop lymphovascular invasion and metastasize to the para-aortic lymphatic chain in a pattern similar to that seen in patients. The extent of (lymph node) metastases is particularly apparent when the implanted tumor cells are transfected to express a fluorescent marker, such as DsRed, which allows the exposed retroperitoneum to be examined by fluorescence microscopy. Described in this unit is a surgical technique for orthotopic implantation and the use of this model for investigating the effects of novel agents as inhibitors of tumor growth and metastasis.

Molecular evidences for the chemosensitizing efficacy of liposomal curcumin in paclitaxel chemotherapy in mouse models of cervical cancer

The microtubule-targeting antineoplastic agent, paclitaxel, is highly efficacious against a wide spectrum of human cancers. However, dose-limiting toxicity and development of drug resistance limit its clinical application. Development of novel strategies that overcome chemoresistance and sensitize cancer cells to paclitaxel can enhance the therapeutic effect of this drug. We have previously shown that curcumin, a natural polyphenol, enhances paclitaxel-induced cytotoxicity in vitro through downregulation of nuclear factor (NF)-κB and Akt pathways. This study was undertaken to determine whether this synergism exists in vivo and to elucidate the underlying molecular mechanisms. Mouse cervical multistage squamous cell carcinoma model using 3-methylcholanthrene (3-MC) and a xenograft model of human cervical cancer in nonobese diabetic severe combined immunodeficient (NOD-SCID) mice using HeLa cells were used to evaluate the synergism. We observed that the combined treatment of curcumin and paclitaxel induced a synergestic reduction in the tumor incidence as well as tumor volume of animals compared with the individual treatments of paclitaxel or curcumin, although curcumin alone could not induce any significant effect at the concentration used. The results suggest that a suboptimal concentration of curcumin augments the antitumor action of paclitaxel by downregulating the activation and downstream signaling of antiapoptotic factors and survival signals such as NF-κB, Akt and mitogen-activated protein kinases that have significant roles in proliferation, survival, angiogenesis and metastasis. This study revealed for the first time that 3-MC-induced tumorigenesis in mice is associated with a strong constitutive activation of NF-κB activity. Furthermore, we also observed that pre-exposure of carcinoma cells isolated from 3-MC-induced tumors to curcumin potentiates paclitaxel-induced apoptosis. Overall, the findings of this preclinical study provide a strong rationale for the validation of this combination through clinical trials. As curcumin could effectively downregulate all these survival signals induced by paclitaxel, we suggest it as a potent chemosensitizer to improve the therapeutic index of paclitaxel.

P38 Kinase Is Crucial for Osteopontin-Induced Furin Expression That Supports Cervical Cancer Progression

p38 kinases activated by growth factors, hormones, and environmental stresses exert diverse functions in regulating normal and malignant cell pathophysiology. Enhanced levels of activated p38 isoforms have been linked with poor prognosis in breast cancer, although the mechanistic basis for this association is poorly understood. In this study, we report that p38 activation in cervical cancer cells is driven by osteopontin (OPN), an extracellular matrix-associated cytokine that drives invasive progression. OPN regulates CD44-mediated p38 phosphorylation that induces NF-κB activation and NF-κB-dependent expression of furin, an extracellular protease implicated in human papilloma virus (HPV) processing that enhances cervical cancer cell motility. OPN induces CD44-mediated MKK3/6 phosphorylation which in turn phosphorylates p38 in these cells. OPN-induced furin expression and cell motility was impeded by blockades to MKK3/6, p38α/β or NF-κB signaling. In a mouse xenograft model of human cervical cancer, tumor growth was enhanced by OPN overexpression and blocked by short hairpin RNA (shRNA)-mediated OPN silencing. Furin overexpression similarly augmented tumor growth in the model, whereas blocking MKK3/6, p38, or furin reduced OPN-induced cervical tumor growth. Analysis of clinical specimens revealed that enhanced expression of OPN, phosphorylated NF-κB, p65, and furin correlated with cervical cancer progression, further strengthening the in vitro and in vivo results. In summary, our findings offer a proof of concept for targeting OPN and its downstream p38 signaling as a novel therapeutic strategy to manage cervical cancer.

Small interfering RNA‐mediated down‐regulation of SPAG9 inhibits cervical tumor growth

The expression of the SPAG9 is associated with various human malignancies. Earlier work revealed a significant association of SPAG9 expression with the early spread of cervical cancer, making it an attractive therapeutic target. Here, the authors investigated the role of SPAG9 in carcinogenesis of squamous cell carcinoma (SCC) of the cervix. Furthermore, they sought to determine whether ablation of SPAG9 expression reduces the tumor growth of cervical SCC in vivo. A plasmid-based small interfering RNA approach was used to specifically knock down the expression of SPAG9 in SiHa cells derived from SCC of the cervix in vitro and in vivo. Reverse transcriptase polymerase chain reaction, immunofluorescence staining, flow cytometry, cellular growth, colony formation, migration, invasion, and wound healing assays were studied to characterize SPAG9 in vitro. Furthermore, a cervical cancer xenograft model in nude mice was established to investigate whether knockdown of SPAG9 reduces the tumor growth of cervical SCC in vivo. The results demonstrated that silencing the SPAG9 by small interfering RNA resulted in inhibition of cell growth, colony formation, migration, and invasion. The authors showed for the first time that the knockdown of SPAG9 expression by small interfering RNA significantly suppressed the tumor growth of cervical SCC in vivo. These results suggest that SPAG9 expression may play a pivotal role in tumor growth and could contribute to the early spread of cervical cancer. Small interfering RNA-mediated down-regulation of SPAG9 represents a promising therapeutic approach for the treatment of cervical cancer.

Thalidomide and angiostatin inhibit tumor growth in a murine xenograft model of human cervical cancer

Objective To determine the impact of thalidomide and angiostatin on tumor growth, angiogenesis, and apoptosis in a xenograft model of cervical cancer. Methods Human umbilical endothelial cells were treated with angiostatin or thalidomide and bFGF-induced proliferation was assessed with the MTT assay. Human cervical cancer cells (CaSki and SiHa) were injected into the flanks of nude mice. After tumors developed, mice were treated with angiostatin 20 mg/kg/day or thalidomide 200 mg/kg/day for 30 days. Fractional tumor growth was determined and immunohistochemical analysis of tumors was used to determine degree of angiogenesis. TUNEL assay was used to assess apoptosis. Results Angiostatin inhibited endothelial cell proliferation by 50–60%. Thalidomide had no direct effect on endothelial cells. Angiostatin and thalidomide both inhibited tumor growth by about 55%. We found no additive or synergistic effect when the two agents were combined. Both agents inhibited angiogenesis and induced apoptosis when compared to tumors from control animals. Conclusions Angiostatin and thalidomide inhibit tumor growth, angiogenesis, and induce apoptosis in this xenograft model of cervical cancer.

551. Enhanced Cell Killing Effect of E1 Modified Adenoviruses Expressing HSV-TK Suicide Gene

Recent clinical trials with Herpes Simplex Virus Thymidine Kinase (HSV-TK) gene therapy using E1-deleted replication-incompetent adenovirus have shown encouraging results, but with limited efficacy. We have previously demonstrated that the cytolytic potency of replication-competent adenoviruses with different combination of E1B genes differed significantly depending on the presence or deletion of E1B 55kDa and E1B 19kDa function (Kim JS. et al. Cancer Gene Therapy). To combine the advantages of the suicide gene approach with that of replicating adenoviruses, we have developed replicating adenoviral vectors harboring HSV-TK, generating Ad-|[Delta]|E1B19/55-TK, Ad-|[Delta]|E1B19-TK, and Ad-|[Delta]|E1B55-TK adenoviruses. The cytotoxicity of the combination therapy with replicating adenoviruses (Ad-|[Delta]|E1B55-TK or Ad-|[Delta]|E1B19/55-TK) and GCV was dramatically increased compared with replicating adenoviruses alone or replication-incompetent adenovirus expressing HSV-TK (Ad-|[Delta]|E1-TK) with GCV. In contrast, the cytotoxicity of the combination therapy with Ad-|[Delta]|E1B19-TK and GCV was significantly reduced when compared to the treatment with Ad-|[Delta]|E1B19-TK alone. In addition, the total viral production of Ad-|[Delta]|E1B19-TK in the presence of GCV was significantly reduced. Taken together, these results suggest that the inhibition of viral replication by the HSV-TK/GCV system counterbalance the cytotoxicity of Ad-|[Delta]|E1B19-TK. The human cervical cancer (C33A) and lung cancer (A549) xenograft models treated with replication-competent adenoviruses with GCV had more significant reduction in tumor growth compared to animals that were injected with PBS or Ad-|[Delta]|E1-TK with GCV. In summary, these data indicate that the suicide gene approach with replicating adenoviruses elicits more enhanced anti-tumor effect than that with replication-incompetent adenovirus.