Cervical Cancer Xenograft Model Research Articles

Orthotopic Xenograft Mouse Model of Cervical Cancer for Studying the Role of MicroRNA-21 in Promoting Lymph Node Metastasis.

Cervical cancer is the most frequent cause of gynecologic cancer-associated death worldwide. Animal models that demonstrate metastatic patterns consistent with the clinical course of cervical cancer are urgently needed to conduct studies focused on understanding the mechanisms of the disease and identifying optimal treatments. To address this, we established an orthotopic xenograft model of cervical cancer in female NOD-SCID mice using SiHa and ME180 cell lines stably expressing green fluorescent protein to evaluate the role of microRNA-21 (miR-21) in spontaneous lymph node metastasis in vivo. In this case, SiHa and ME180 cells were transduced by lentivirus to stably express green fluorescent protein and miR-21. Overexpression of miR-21 promoted proliferation, migration, and invasion of SiHa and ME180 cells in vitro. Finally, an orthotopic xenograft model of human cervical cancer was successfully established in NOD-SCID mice. Using this model, we confirmed that overexpression of miR-21 resulted in an increase in the size of primary tumors and in the frequency of spontaneous lymph node metastasis at the time of excision. Therefore, the use of the orthotopic xenograft model should allow for the investigation of novel factors that affect metastasis of cervical cancer and presents an opportunity to evaluate potential therapeutic agents that may inhibit the spread of the disease.

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.

Alpha-mangostin induces apoptosis through activation of reactive oxygen species and ASK1/p38 signaling pathway in cervical cancer cells.

Alpha-mangostin, a natural xanthonoid, has been reported to possess the anti-cancer property in various types of human cancer. However, its effects and mechanism of α-mangostin in cervical cancer remain unclear. We found that α-mangostin effectively inhibited cell viability, resulted in loss of mitochondrial membrane potential (MMP), release of cytochrome C, increase of Bax, decrease of Bcl-2, and activation of caspase-9/caspase-3 cascade in cervical cancer cells. Alpha-mangostin elevated the contents of reactive oxygen species (ROS) to activate p38. Disrupting ASK1/p38 signaling pathway by a specific inhibitor of p38, or by the siRNAs against ASK1, MKK3/6, or p38, significantly abolished α-mangostin-induced cell death and apoptotic responses. Moreover, α-mangostin also repressed tumor growth in accordance with increased levels of p-ASK1, p-p38, cleaved-PARP and cleaved-caspase-3 in the tumor mass from the mouse xenograft model of cervical cancer. In the current study, we provided first evidence to demonstrate that dietary antioxidant α-mangostin could inhibit the tumor growth of cervical cancer cells through enhancing ROS amounts to activate ASK1/p38 signaling pathway and damage the integrity of mitochondria and thereby induction of apoptosis in cervical cancer cells.

Cervical Cancer Growth Is Regulated by a c-ABL-PLK1 Signaling Axis.

The nonreceptor tyrosine kinase c-ABL controls cell growth but its contributions in solid tumors are not fully understood. Here we report that the Polo-like kinase PLK1, an essential mitotic kinase regulator, is an important downstream effector of c-ABL in regulating the growth of cervical cancer. c-ABL interacted with and phosphorylated PLK1. Phosphorylation of PLK1 by c-ABL inhibited PLK1 ubiquitination and degradation and enhanced its activity, leading to cell-cycle progression and tumor growth. Both c-ABL and PLK1 were overexpressed in cervical carcinoma. Notably, PLK1 tyrosine phosphorylation correlated with patient survival in cervical cancer. In a murine xenograft model of human cervical cancer, combination treatment with c-ABL and PLK1 inhibitors yielded additive effects on tumor growth inhibition. Our findings highlight the c-ABL-PLK1 axis as a novel prognostic marker and treatment target for human cervical cancers. Cancer Res; 77(5); 1142-54. ©2016 AACR.

Hedgehog inhibition enhances efficacy of radiation and cisplatin in orthotopic cervical cancer xenografts

Background:The Hedgehog (Hh) pathway is upregulated in cervical cancer and associated with poor outcome. We explored the effects of Hh pathway inhibition in combination with RTCT in a patient derived orthotopic cervical cancer xenograft model (OCICx).Methods:5E1, a monoclonal antibody for SHH, or Sonidegib (LDE225), a clinical SMO inhibitor (Novartis) were added to RTCT. We investigated tumour growth delay, metastasis and GI toxicity using orthotopic cervical cancer xenografts models. The xenografts were treated with radiotherapy (15 × 2 Gy daily fractions over 3 weeks) and weekly cisplatin 4 mg kg−1 concurrently, with or without 5E1 or Sonidegib (LDE225). The Hh inhibitors were administered by subcutaneous injection (5E1; 20 mg kg−1 weekly for 3 weeks), or by oral gavage (Sonidegib; 60 mg kg−1 daily for 3 weeks).Results:We observed that both Hh inhibitors administered with RTCT were well tolerated and showed increased tumour growth delay, and reduced metastasis, with no increase in acute GI-toxicity relative to RTCT alone.Conclusions:Our data suggest Hh can be a valid therapeutic target in cervical cancer and supports data suggesting a potential therapeutic role for targeting Hh in patients undergoing RTCT. This warrants further investigation in clinical trials.

In Vitro and In Vivo Synergistic Therapeutic Effect of Cisplatin with Human Papillomavirus16 E6/E7 CRISPR/Cas9 on Cervical Cancer Cell Line

PURPOSE: Human papillomavirus (HPV) type 16 is one of the major etiologic factors of cervical cancer. Our study aims to investigate the potentiality of the antiviral clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated Cas9 system (CRISPR/Cas9) targeting the E6 and E7 oncogenes of HPV16 as a potential chemosensitizer of cisplatin (cis-diaminedichloroplatinum II; CDDP) for cervical cancer. METHODS: Specifically, the therapeutic efficacy of combination of CDDP and HPV16 E6 + E7-CRISPR/Cas9 was assessed in cervical cancer cells and cervical cancer xenograft models. RESULTS: In vitro experiments showed that long-term exposure of SiHa cells to the HPV16 E6 + E7-CRISPR/Cas9 induced apoptosis, and its pro-apoptosis effect became more obvious when combined with CDDP. In vivo study found the efficacy of the combination of HPV16 E6 + E7-CRISPR/Cas9 and CDDP were superior to either of the treatments in term of apoptosis induction and metastasis inhibition. CONCLUSION: Collectively, our results suggested that HPV16 E6 + E7-CRISPR/Cas9 could be an effective sensitizer of CDDP chemotherapy in cervical cancer.

Use of rhenium-188 for in vivo imaging and treatment of human cervical cancer cells transfected with lentivirus expressing sodium iodide symporter.

Although survival rates for cervical cancer have improved, they need further improvement in patients with distant metastases. The sodium iodine symporter (NIS) gene has often been used in cancer therapy and imaging. We examined the therapeutic effects of rhenium-188 (188Re) in a cervical cancer xenograft model expressing the NIS gene under the control of the tumor-specific human telomerase reverse transcriptase (hTERT) promoter. We constructed two recombinant lentiviral vectors expressing enhanced green fluorescent protein (eGFP) or the NIS gene driven by the hTERT promoter. To determine the tumor-specific transcriptional activity of the hTERT promoter, the eGFP-expressing vector was stably transfected into tumor cells and normal cells. A cervical cancer HeLa cell line stably expressing NIS (HeLa-TERTNIS) was created and examined in a similar way. HeLa and HeLa-TERTNIS tumor xenografts were transplanted in nude mice, and invivo 188Re distribution was measured using micro-SPECT/CT imaging. The therapeutic effects of 188Re were assessed over 21 days on the basis of tumor volume and the immunohistochemical findings of excised tumors. eGFP expression controlled by the hTERT promoter was substantially higher in the tumor cells than normal cells. Quantitative PCR and western blotting confirmed that HeLa-TERTNIS cells expressed high levels of NIS mRNA and protein, respectively. Further, 188Re uptake and accumulation were significantly higher in HeLa-TERTNIS cells and xenografts than HeLa cells and xenografts. Invitro and invivo, 188Re significantly reduced the survival of HeLa-TERTNIS cells and inhibited the growth of HeLa-TERTNIS xenografts, respectively. Immunohistochemical staining showed that HeLa-TERTNIS xenograft tumors expressed higher levels of NIS and caspase-3 and lower levels of Ki-67 than HeLa xenograft tumors. Our findings indicated that hTERT promoter-driven expression of the NIS gene in HeLa cells led to 188Re uptake and therapeutic effects. Thus, NIS-based gene therapy and imaging using the hTERT promoter and 188Re may be possible.

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.

The effects of a picosecond pulsed electric field on angiogenesis in the cervical cancer xenograft models

ObjectiveThe application of picosecond pulsed electric field (psPEF) is a new biomedical engineering technique used in cancer therapy. However, its effects on cervical cancer angiogenesis are not clear. Therefore, the aim of the present study is to investigate the effects of psPEF on angiogenesis in cervical cancer xenograft models. MethodsXenograft tumors were created by subcutaneously inoculating nude mice (athymic BALB/c nu/nu mice) with HeLa cells, then were placed closely between tweezer-type plate electrodes and subjected to psPEF with a gradually increased electric field intensity (0kV/cm, 50kV/cm, 60kV/cm, 70kV/cm). The direct effect on tumor tissue was observed by hematoxylin and eosin (H&E) staining and transmission electron microscopy (TEM). The changes of blood vessels and oxygen saturation (sO2) of tumors were monitored in vivo by photoacoustic tomography (PAT). The microvessel density (MVD), vascular endothelial growth factor (VEGF) and hypoxia-inducible transcription factors (HIF-1α and HIF-2α) were detected by immunohistochemical technique (IHC). Their protein expressions and gene transcription levels were evaluated using western blot (WB) and quantitative reverse transcription and polymerase chain reaction (RT-PCR). ResultsPsPEF induced obvious necrosis of cervical cancer tissue; with the increasing of electric field intensity, the MVD, vascular PA signal and sO2 values declined significantly. The protein expression and gene transcription levels of VEGF, HIF1α and HIF2α were significantly decreased at the same time. ConclusionPsPEF exhibited dramatic anti-tumor and anti-angiogenesis effects in cervical cancer xenograft models by exerting direct effect on cancer cells and vascular endothelial cells and indirect effect on tumor angiogenesis-related factors.

Ultrasound-responsive microbubbles for sonography-guided siRNA delivery

RNA interfering is a gene therapeutic approach of great potential for cancer. However, tumor-targeted delivery of small interfering RNA (siRNA) solely based on the enhanced permeability and retention effect of nanocarriers is often insufficient. To address this challenge, siRNA encapsulated ultrasound-responsive microbubble (MB) was developed from polymeric siRNA micelles and liposomal MBs using hetero-assembling strategy. 1MHz low-frequency ultrasound exposure of the tumor site after intratumoral injection of XIAP siRNA/MBs led to enhanced permeability for much more siRNA delivery into deep tumor regions. Significant improvement of XIAP gene silencing and cleaved caspase-3 activation was achieved, resulting in good therapeutic effect on human cervical cancer xenograft model in nude mice. Moreover, real-time US monitoring of the tumor was also possible using the siRNA/MBs as a contrast agent during the therapeutic process. These results show that the multi-functional siRNA/MBs are a promising theranostic system for cancer gene therapy.

Gadolinium‐Doped Iron Oxide Nanoprobe as Multifunctional Bioimaging Agent and Drug Delivery System

In this study, a high‐performance T1–T2 dual‐model contrast agent by gadolinium‐doped iron oxide nanoparticle (GION) is developed. Following its development, the application of this agent in vivo by combining doxorubicin (DOX) and folic acid (FA) (FA–GION–DOX) for targeted drug delivery to monitor cancer treatment is explored. GION showed transverse and longitudinal relaxivities up to 182.7 × 10−3 and 7.87 × 10−3m−1 s−1, respectively, upon Gd/Fe ratio in GION at 1/4. DOX released from FA–GION–DOX is pH dependent and only kills cancer cell after FA receptor‐mediated internalization into the acidic environment of endosomes and lysosomes. Systemic delivery of FA–GION–DOX significantly inhibits the growth of tumors and shows good magnetic resonance enhancement in a human cervical cancer xenograft model. Thus, FA–GION–DOX has a potential application for the targeted and magnetic resonance imaging guided therapy of cervical cancer.

Porphine functionalized nanoparticles of star-shaped poly(ε-caprolactone)-b-D-α-tocopheryl polyethylene glycol 1000 succinate biodegradable copolymer for chemophotodynamic therapy on cervical cancer

We developed a system of biodegradable nanoparticles (NPs) of 5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphine (TAPP) centered, 4 arm star-shaped copolymers based on poly(ε-caprolactone) (PCL) and D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) for combinatory chemophotodynamic therapy by using docetaxel (DTX) as a model anticancer drug and TAPP as photodynamic sensitizer. TPGS component in the copolymer plays an important role in enhancing the drug encapsulation efficiency, drug release kinetics and cellular uptake of the NPs, as well as in overcoming the multidrug resistance due to inhibition of P-glycoproteins (P-gp) of the cancer cells. We demonstrated in vitro by using the MCF7/ADR breast cancer cells of P-gp overexpression and the HeLa cervical cancer cells that the proposed chemophotodynamic therapy by the DTX-loaded TAPP-PCL-b-TPGS NPs could have much higher therapeutic effect than the original drug Taxotere®. IC50 data showed that the DTX-loaded TAPP-PCL-b-TPGS NPs chemophotodynamic therapy could be 9.36 and 56.5-fold efficient after 24 and 48h treatment, respectively in comparison with the Taxotere® chemotherapy. The in vivo investigation by employing a cervical cancer xenograft model further confirmed the advantages of the proposed chemophotodynamic therapy by the DTX-loaded TAPP-PCL-b-TPGS NPs versus the Taxotere® chemotherapy.

Sphingosine kinase 1 is a reliable prognostic factor and a novel therapeutic target for uterine cervical cancer.

Sphingosine kinase 1 (SPHK1), an oncogenic kinase, has previously been found to be upregulated in various types of human malignancy and to play a crucial role in tumor development and progression. Although SPHK1 has gained increasing prominence as an important enzyme in cancer biology, its potential as a predictive biomarker and a therapeutic target in cervical cancer remains unknown. SPHK1 expression was examined in 287 formalin-fixed, paraffin-embedded cervical cancer tissues using immunohistochemistry, and its clinical implications and prognostic significance were analyzed. Cervical cancer cell lines including HeLa and SiHa were treated with the SPHK inhibitors SKI-II or FTY720, and effects on cell survival, apoptosis, angiogenesis, and invasion were examined. Moreover, the effects of FTY720 on tumor growth were evaluated using a patient-derived xenograft (PDX) model of cervical cancer. Immunohistochemical analysis revealed that expression of SPHK1 was significantly increased in cervical cancer compared with normal tissues. SPHK1 expression was significantly associated with tumor size, invasion depth, FIGO stage, lymph node metastasis, and lymphovascular invasion. Patients with high SPHK1 expression had lower overall survival and recurrence-free survival rates than those with low expression. Treatment with SPHK inhibitors significantly reduced viability and increased apoptosis in cervical cancer cells. Furthermore, FTY720 significantly decreased in vivo tumor weight in the PDX model of cervical cancer. We provide the first convincing evidence that SPHK1 is involved in tumor development and progression of cervical cancer. Our data suggest that SPHK1 might be a potential prognostic marker and therapeutic target for the treatment of cervical cancer.

Combination of IL-24 and cisplatin inhibits angiogenesis and lymphangiogenesis of cervical cancer xenografts in a nude mouse model by inhibiting VEGF, VEGF-C and PDGF-B.

The aim of the present study was to investigate the synergistic inhibitory effects of recombinant IL-24 delivered by pDC316-hIL-24 transfection and cisplatin (DDP) on angiogenesis and lymphangiogenesis in a cervical cancer xenograft model established in nude mice. Thirty-six mice (successful model) were randomly divided into six groups (n=6 in each): i)phosphate-buffered saline control; ii) empty plasmid; iii)half-dose DDP; iv) recombinant interleukin (IL)-24; v)full-dose DDP; and vi) combined treatment. Tumor growth and animal weight were measured every 3 days. Animals were sacrificed by cervical dislocation at 2 weeks after the cessation of treatment. Tumor inhibition was compared after intervention. Lymph node metastasis was evaluated by immunohistochemical (IHC) analysis of vascular endothelial growth factor (VEGF), VEGF-C and VEGFR-3. Platelet-derived growth factor (PDGF)-B expression was also investigated by western blot analysis. Microvessel density was evaluated by IHC analysis of CD34 expression. The tumor growth was slower or reduced in the IL-24 and half-dose DDP+IL-24 groups. The expression of VEGF and microvessel density in the IL-24 group was significantly lower than that in the other groups. VEGF (VEGF-A), VEGF-C, VEGFR-3 and PDGF-B expression was significantly decreased in the IL-24 and half-dose DDP+IL-24 groups compared with that in the other groups (P<0.001). The recombinant plasmid pDC316-hIL-24 acts synergistically with cisplatin to inhibit tumor growth and angiogenesis. Our data indicate that these effects are mediated by downregulation of VEGF, VEGF-C and PDGF-B expression. Thus, IL-24 may enhance tumor chemosensitivity to cisplatin, which may be an important strategy for reducing the side-effects of this chemotherapy.

Abstract 1204: Establishment of a patient-derived orthotopic xenograft (PDOX) model of patient cervical cancer

Abstract We implanted a tumor resected from a patient with metastatic HER-2-positive cervical cancer from 8 NOD/SCID mice (F1; NOD/SCID-sc) and 10 nude mice (F1; Nude-sc) subcutaneously, and 8 nude mice (F1; Nude-soi) orthotopically. Five out of 8 NOD/SCID mice died within 7 days after implantation. Infections around the surgical wound, loss of weight and of lethargy were observed in post-operatively in other mice. We examined the tumor-take rate of each model 8 weeks after implantation. Tumors grew in 3 out of 3 mice in F1; NOD/SCID-sc (100 %); 7 out of 10 mice in F1; nude-sc (70 %) and 6 out of 8 mice in F1; nude-soi group (75 %), respectively. The metastatic tumors (peritoneal dissemination, liver metastasis, lung metastasis or lymph node metastasis) were detected in 4 mice in F1; nude-soi mice (50 %). We found all xenografts including the metastatic tumors had histological structures similar to the original tumor and were stained by anti-human HER-2 antibody. Next, we implanted the small fragments of subcutaneous tumors from F1; Nude-sc mice (F2; sc-sc) or the primary tumors of F1; nude-soi mice (F2; soi-sc) to another set of nude mice (20 mice each) subcutaneously, and examined the tumor-take rate of each group 4 weeks and 8 weeks after implantation. Eight mice in the F2; sc-sc group (40 %) and 16 mice in the F2; soi-sc group (80 %) had tumors at week 4, and 15 mice in the F2; sc-sc group (75 %) and 19 mice in the F2; soi-sc group (95 %) had tumors at week 8. These results suggest that PDOX in nude mouse model recapitulates the biological behaviors of the original tumor and has the sufficient tumor-take rate. 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, Ali Maawy, Itaru Endo, Robert M. Hoffman. Establishment of a patient-derived orthotopic xenograft (PDOX) model of patient cervical cancer. [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 1204. doi:10.1158/1538-7445.AM2014-1204

Hedgehog signalling in primary cervix xenograft (OCICx) models: A potential new therapeutic target in combination with chemoradiotherapy.

11120 Background: Overcoming treatment resistance and repopulation in patients undergoing chemo-radiotherapy for primary treatment of cervical cancer is critical in order to improve long-term survival. Upregulation of Hh gene expression in women with cervical cancer prior to chemoradiotherapy is associated with a worse outcome. Aim: to investigate inhibition of the Hh pathway in combination with chemoradiotherapy in orthotopic primary cervical cancer xenograft models (OCICX). Methods: mRNA expression levels of Hh pathway members were characterized using qRT-PCR analysis in OCICx models. To simulate clinical treatment 5E1 (antibody blocking SHH and IHH ligand interaction with PTCH1) was administered to mice bearing cervix xenografts (20 mg/kg/weekly) with localized fractionated irradiation (15 x 2Gy fractions given 5 days/wk over 3 wks). Tumor growth delay, lymph node metastases, survival and toxicity were assessed. Results: The OCICX models exhibited matched levels of Hh gene expression compared to the pa...

Targeting of Histone Deacetylases to Reactivate Tumour Suppressor Genes and Its Therapeutic Potential in a Human Cervical Cancer Xenograft Model

Aberrant histone acetylation plays an essential role in the neoplastic process via the epigenetic silencing of tumour suppressor genes (TSGs); therefore, the inhibition of histone deacetylases (HDAC) has become a promising target in cancer therapeutics. To investigate the correlation of histone acetylation with clinicopathological features and TSG expression, we examined the expression of acetylated H3 (AcH3), RARβ2, E-cadherin, and β-catenin by immunohistochemistry in 65 cervical squamous cell carcinoma patients. The results revealed that the absence of AcH3 was directly associated with poor histological differentiation and nodal metastasis as well as reduced/negative expression of RARβ2, E-cadherin, and β-catenin in clinical tumour samples. We further demonstrated that the clinically available HDAC inhibitors valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA), in combination with all-trans retinoic acid (ATRA), can overcome the epigenetic barriers to transcription of RARβ2 in human cervical cancer cells. Chromatin immunoprecipitation analysis showed that the combination treatment increased the enrichment of acetylated histone in the RARβ2-RARE promoter region. In view of these findings, we evaluated the antitumor effects induced by combined VPA and ATRA treatment in a xenograft model implanted with poorly differentiated human squamous cell carcinoma. Notably, VPA restored RARβ2 expression via epigenetic modulation. Additive antitumour effects were produced in tumour xenografts by combining VPA with ATRA treatment. Mechanistically, the combination treatment reactivated the expression of TSGs RARβ2, E-cadherin, P21CIP1, and P53 and reduced the level of p-Stat3. Sequentially, upregulation of involucrin and loricrin, which indicate terminal differentiation, strongly contributed to tumour growth inhibition along with partial apoptosis. In conclusion, targeted therapy with HDAC inhibitors and RARβ2 agonists may represent a novel therapeutic approach for patients with cervical squamous cell carcinoma.

Gene silencing of A-kinase anchor protein 4 inhibits cervical cancer growth in vitro and in vivo

Earlier, we reported an association of A-kinase anchor protein 4 (AKAP4) expression in cervical cancer patient specimens, indicating its implications as an immunotherapeutic target. In this study, we investigated the possible role of AKAP4 in cervical carcinogenesis. AKAP4 messenger RNA and protein expression was assessed in four cervical cancer cell line models, C-33A, CaSki, HeLa and SiHa. Gene silencing approach was employed to investigate the potential role of AKAP4 in cellular growth, proliferation, colony-forming ability, migration and invasion in aggressive squamous cell carcinoma cells (SiHa). Further, the effect of downregulation of AKAP4 on tumor growth was examined in the cervical cancer xenograft model in nude mice. Our data clearly indicated that AKAP4 was expressed in all cervical cancer cells at the gene and protein level. We also observed distinct cytoplasmic and surface localization by indirect immunofluorescence and flow cytometry, respectively. Ablation of AKAP4 protein caused significant inhibition in cellular proliferation, colony-forming ability, migration and invasion ability of SiHa cells. Further, gene silencing of AKAP4 also resulted in reduced tumor growth in nude mice in vivo. Collectively, AKAP4 surface localization and its significant association with malignant properties of cervical cancer cells imply its clinical utility as an immunotherapeutic target.

Abstract 3437: P276, a cyclin-dependent kinase inhibitor with antitumor activity against high-risk human cervical cancer.

Abstract Cervical cancer is the third most common cancer worldwide in women. While preventive vaccines have been approved for cervical cancer, treatment options are limited to chemotherapy drugs. An unmet need, thus, exists for the treatment of cervical cancer. High-risk human papilloma viruses (HPVs), type 16 and 18, account for 70% of the cervical cancer. Viral oncoproteins E6 and E7 bind and inhibit the p53 and Rb tumor suppressors, respectively. One attractive strategy for the treatment of cervical cancer is to attenuate the expression of E6 and E7 viral oncoproteins by a pharmacological inhibitor of cyclin-dependent kinases (CDKs). P276 is a CDKs inhibitor with potent activity against CDK4, CDK1 and CDK9 (Joshi et al, Mol. Cancer Ther. 2007; 6:918-25). The therapeutic potential and mechanism of action of P276 in cervical cancer was investigated using high-risk HPV type 16 (SiHa and Caski) and type 18 (HeLa) human cervical cancer cell line. P276 inhibited cell growth with sub-micromolar IC50 values (0.5-0.8 μM), induced cell cycle arrest and apoptosis in cervical cancer lines. E6 negatively regulates p53 protein stability in HPV-infected cervical cancer. P276 downregulated mRNA and protein expression of E6 in SiHa cells with concomitant upregulation of p53 protein and its downstream target p21, indicating activation of the p53 pathway. It also downregulated E7 mRNA expression in SiHa and HeLa cell lines. Further, it is known that hyperphosphorylation of Rb relieves its interaction with E2F, leading to Rb inactivation and E2F activation. Treatment with P276 dephosphorylated Rb at Ser780, indicating Rb activation. P276 demonstrated significant (p&amp;lt;0.01) in vivo efficacy in HPV type 16 SiHa human cervical cancer xenograft model when administered by i.p. route, Treatment with 25 mg/kg of P276 for 21 days induced 59% of tumor growth inhibition without body weight loss in this model. Conclusion: P276, a CDKs inhibitor, is efficacious against high-risk cervical cancer. Citation Format: Sanjeev Kumar Shangary, Gandhali Deshpande, Vinay Sonawane, Venkatesha Venkatasubbaiah, Archana Jalota-Badhwar, Asavari Joshi, Manoj Mayekar, Nitesh Shirsath, Prashant Pandey, Veena R. Agarwal. P276, a cyclin-dependent kinase inhibitor with antitumor activity against high-risk human cervical cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3437. doi:10.1158/1538-7445.AM2013-3437

Comparisons of 5-aminolevulinic acid photodynamic therapy and after-loading radiotherapy in vivo in cervical cancer

To compare the differences between 5-aminolevulinic acid photodynamic therapy (5-ALA-PDT) with traditional after-loading radiotherapy in aspects of efficacies and side effects. MTT assay was adopted to detect the inhibitive effects of 5-ALA-PDT on Hela cells proliferation. Flow cytometry was used to analyze cell apoptosis. After establishment of human cervical cancer xenograft model, the comparisons between 5-ALA-PDT with radiotherapy were performed with respect to treatment efficacies (survival rate, body weight, and tumor volume) and side effects (appearance and behavior, ovarian endocrine functions, and skin lesion around the tumor). 5-Aminolevulinic acid photodynamic therapy exerted killing effects on cervical cancer cells. Morphological changes and flow cytometric analyses indicated apoptosis to be one of the mechanisms for tumor growth suppression. Both proliferation inhibition and cell apoptosis showed dependency on photosensitizer concentration and irradiation intensity. Repeated photodynamic therapy presented stronger inhibitive effects on tumor growth compared to after-loading radiotherapy, while producing milder impairment of ovarian endocrine functions and skin lesions around the tumors. 5-Aminolevulinic acid photodynamic therapy has great potential to be an alternative treatment modality for cervical cancer.