Cell Carcinoma Of Cervix Research Articles

Small cell carcinoma of the uterine cervix in a pregnant patient diagnosed with liquid based cytology and cell block immunocytochemistry.

Definitive cytomorphologic diagnosis of small cell carcinoma of the uterine cervix is possible but can be challenging in routine cervicovaginal cancer screening specimens. Several small series of reported cases of cervical small cell carcinoma have shown this uncommon malignancy to represent fewer than 2% of all invasive cervical cancers. This tumor type is associated with poor prognosis and rapid disease progression and can develop to an advanced stage in the interval between screening visits. Only rare case reports of small cell carcinoma arising in gravid cervices are known. In the current case a 29-year-old, gravida 6, para 2, pregnant (10-week gestation) female presented with postcoital bleeding. A definitive diagnosis of small cell carcinoma of the cervix was made possible by liquid based Pap testing with ancillary cell block preparation allowing for immunocytochemical characterization of the lesional cell population.

Paraneoplastic dermatomyositis with carcinoma cervix: a rare clinical association.

Dermatomyositis is an uncommon inflammatory myopathy associated with cutaneous manifestations. It may also occur as paraneoplastic syndrome associated with various malignancies, most common of which being lung, breast, stomach, rectum, kidney, or testicular cancer. A postmenopausal woman presented to us with generalized itching along with skin rash and proximal muscle weakness of 2 years' duration. Examination revealed heliotrope rash and mechanic hands and muscle power 2/5 in proximal muscle groups of both upper and lower limbs. A clinical diagnosis of dermatomyositis was made which was supported by raised lactate dehydrogenase levels and skin biopsy findings. Past history was significant for vaginal discharge and bleeding per vagina. Further work-up revealed carcinoma cervix and she was referred to oncology department for further management. Temporal relationship and improvement of muscle weakness with treatment of underlying neoplasm supported its paraneoplastic nature. So, final diagnosis of keratinizing squamous cell carcinoma of cervix with paraneoplastic dermatomyositis was made. A nationwide cohort study of 1,012 patients with dermatomyositis in Taiwan revealed only 3 patients with cervical cancer. So this case is being reported for its rare association with carcinoma cervix and to highlight the need of detailed evaluation for underlying malignancies in patients with dermatomyositis.

Composition of lipoidal matter and evaluation of hepatoprotective, cytotoxic, and antioxidant activities of Khaya grandifoliola C.DC. growing in Egypt

Background and objectives Liver disease is one of the most common health problems. Schistosoma and hepatic viruses are widespread in Egypt, causing severe liver damage and posing a threat to life, which has motivated researchers to discover and evaluate new hepatoprotective agents, particularly from natural sources. The hepatoprotective effect of the leaf extracts of Khaya grandifoliola C.DC. were investigated, after determination of acute toxicity. The anticancer activity and antioxidant potential of the plant were evaluated, as well as the phytochemical composition of petroleum ether and chloroform extracts. Materials and methods Different extracts of K. grandifoliola C.DC. leaves were prepared and tested for hepatoprotective effect against carbon tetrachloride-induced liver damage in rats using silymarin as the reference drug. Anticancer activity was tested on HEPG2 (liver carcinoma cell line), HCT116 (colon carcinoma cell line), HELA (cervix carcinoma cell line), HEP2 (larynx carcinoma cell line), and MCF7 (breast carcinoma cell line). Its potency was compared with the reference drug Doxorubicin. The antioxidant potential was evaluated using 1,1-diphenyl-2-picrylhydrazyl free radical scavenging assay and ascorbic acid as the reference drug. After saponification of the petroleum ether extract, unsaponifiable matter and fatty acid methyl esters were analyzed by GC/MS. The chloroform extract was subjected to vacuum liquid chromatography. Results and conclusion The ethanolic extract of the leaves showed no toxicity up to 5 g/kg. It exhibited potent cytotoxic activity against HCT116 (colon carcinoma cell line) and MCF7 (breast carcinoma cell line), compared with doxorubicin as the standard. Also the ethanolic extract has significant free radical scavenger (100% inhibition) activity, compared with ascorbic acid. The extracts showed significant hepatoprotective and curative activity. GC/MS analysis of both unsaponifiable matter and fatty acids from petroleum ether extract allowed identification of 94.29% of the total unsaponifiable matter, (hentriacontane represented the major component, 11.98%) and 80.72% of the fatty acid methyl ester content (hexadecanoic acid methyl ester represented the major component, 31.68%). Vacuum liquid chromatography of chloroform extract led to isolation of two sterol glucosides (β-sitosterol-3-O-β-d-glucopyranoside and β-stigmasterol-3-O-β-d-glucopyranoside).

Dihydroartemisinine Enhances Dictamnine-induced Apoptosis via a Caspase Dependent Pathway in Human Lung Adenocarcinoma A549 Cells

Dictamnine (Dic) has the ability to exert cytotoxicity in human cervix, colon, and oral carcinoma cells and dihydroartemisinin (DHA) also has potent anticancer activity on various tumour cell lines. This report explores the molecular mechanisms by which Dic treatment and combination treatment with DHA and Dic cause apoptosis in human lung adenocarcinoma A549 cells. Dic treatment induced concentration- and time-dependent cell death. FCM analysis showed that Dic induced S phase cell cycle arrest at low concentration and cell apoptosis at high concentration in which loss of mitochondrial membrane potential (Δψmm) was not involved. In addition, inhibition of caspase-3 using the specific inhibitor, z-DQMD-fmk, did not attenuate Dic-induced apoptosis, implying that Dic-induced caspase-3-independent apoptosis. Combination treatment with DHA and Dic dramatically increased the apoptotic cell death compared to Dic alone. Interestingly, pretreatment with z-DQMD-fmk significantly attenuated DHA and Dic co-induced apoptosis, implying that caspase-3 plays an important role in Dic and DHA co-induced cell apoptosis. Collectively, we found that Dic induced S phase cell cycle arrest at low concentration and cell apoptosis at high concentration in which mitochondria and caspase were not involved and DHA enhanced Dic induced A549 cell apoptosis via a caspase-dependent pathway.

Metastasis from cervical carcinoma presenting with acute intestinal obstruction – a case report

A 55 yr. old woman attended emergency with acute intestinal obstruction. The patient underwent emergency surgical procedure of intestinal resection after straight X-ray,few haematological and biochemical investigations. On gross examination ileal stricture due to mass in ileocaecal region with mesenteric lymph nodes found which on histological examination came out to be a squamous cell carcinoma with metastatic deposit in lymph node. The patient had a hysterectomy done 3&1/2 years back for Stage IIB squamous cell carcinoma of cervix for which she received chemoradiation.The intestinal obstruction very likely to be a metastatic presentation. DOI: http://dx.doi.org/10.3329/bjms.v12i4.16666 Bangladesh Journal of Medical Science Vol. 12 No. 04 October ’13 Page 439-441

Characteristics of quercetin interactions with liposomal and vacuolar membranes

Quercetin (3,3′,4′,5,7-pentahydroxyflavone) is claimed to exert many beneficial health effects. With application of 1H NMR (nuclear magnetic resonance) and FTIR (Fourier-transform infrared) techniques, quercetin interaction with liposomes formed with dipalmitoylphosphatidylcholine (DPPC) was analyzed. Patch-clamp technique was employed to study quercetin effects at single channel level of vacuolar membranes in the liverwort Conocephalum conicum. Light and electron microscopy were applied to study quercetin effects on human negroid cervix carcinoma cells (HeLa). Enzymatic measurements along with DPPH (1,1-diphenyl-2-picrylhydrazyl) bioassay were performed to investigate the influence of quercetin on antioxidant enzymes and reactive oxygen species (ROS) production. The inclusion of quercetin to the membrane exerted pronounced ordering effect on the motional freedom of lipids in the head group region as manifested by broadening of the 1H NMR spectral line representing the choline groups. FTIR analysis revealed quercetin incorporation into DPPC liposomes via hydrogen bonding between its own hydroxyl groups and lipid polar head groups in the C–O–P–O–C segment. Both, FTIR and NMR techniques indicated also quercetin spectral effects in the region corresponding to alkyl chains. Patch-clamp experiments showed that quercetin stabilizes tonoplast and promotes a close state of SV channels. Microscopic observations of HeLa cells revealed characteristic changes in ultrastructure and morphology of the examined cells in comparison to control cells. Pretreatment of HeLa cells with quercetin alleviated H2O2-induced cell injury by improving redox balance as indicated by the increase in glutathione content and SOD (superoxide dismutase) levels as well as by the decrease in ROS level. \\In conclusion, the incorporation, distribution and the changes of biophysical properties of the membranes are very important for the effectiveness of phenolic compounds as antioxidant and anticancer factors.

Targeted expression of human folylpolyglutamate synthase for selective enhancement of methotrexate chemotherapy in osteosarcoma cells

The antifolate methotrexate (MTX) is an important chemotherapeutic agent for treatment of osteosarcoma. This drug is converted intracellularly into polyglutamate derivates by the enzyme folylpolyglutamate synthase (FPGS). MTX polyglutamates show an enhanced and prolonged cytotoxicity in comparison to the monoglutamate. In the present study, we proved the hypothesis that transfer of the human fpgs gene into osteosarcoma cells may augment their MTX sensitivity. For this purpose, we employed the human osteocalcin (OC) promoter, which had shown marked osteosarcoma specificity in promoter studies using different luciferase assays in osteosarcoma and non-osteosarcoma cell lines. A recombinant lentiviral vector was generated with the OC promoter driving the expression of fpgs and the gene for enhanced green fluorescent protein (egfp), which was linked to fpgs by an internal ribosomal entry site (IRES). As the vector backbone contained only a self-inactivating viral LTR promoter, any interference of the OC promoter by unspecific promoter elements was excluded. We tested the expression of FPGS and enhanced green fluorescent protein (EGFP) after lentiviral transduction in various osteosarcoma cell lines (human MG-63 cells and TM 791 cells; rat osteosarcoma (ROS) 17/2.8 cells) and non-osteogenic tumor cell lines (293T human embryonic kidney cells, HeLa human cervix carcinoma cells). EGFP expression and MTX sensitivity were assessed in comparison with non-transduced controls. Whereas the OC promoter failed to enhance MTX sensitivity via FPGS expression in non-osteogenic tumor cell lines, the OC promoter mediated a markedly increased MTX cytotoxicity in all osteosarcoma cell lines after lentiviral transduction. The present chemotherapy-enhancing gene therapy system may have great potential to overcome in future MTX resistance in human osteosarcomas.

In vitro antioxidant and cytotoxic properties of ethanol extract of Alpinia oxyphylla fruits

Context. Alpinia oxyphylla Miquel (Zingiberaceae) is a traditional Chinese herbal medicine widely used for the treatment of intestinal disorders, urosis and diuresis. However, information about antioxidant and cytotoxic properties of its fruits remains to be elucidated.Objective: The ethanol crude extract (CE) and its fractions [petroleum ether fraction (PF), ethyl acetate fraction (EF), n-butanol fraction (BF) and water fraction (WF) extracted by petroleum ether, ethyl acetate, n-butanol and water, respectively] of A. oxyphylla fruits were investigated for their antioxidant activity and cytotoxicity.Materials and methods: The total phenolic content (TPC) and antioxidant activity of the extracts were determined by Folin–Ciocalteu reagent, 1,1-diphenyl-2-picrylhydrazyl (DPPH•), Trolox equivalent antioxidant capacity and reducing power assay. Cytotoxicity of the extracts (0–200 μg/mL) was tested on six human cancer cell lines (breast cancer cell line, cervix carcinoma cell line, lung adenocarcinoma cell line, liver carcinoma cell line, gastric cancer cell line and colon cancer cell line) using the sulforhodamine B assay.Results: The TPC of extracts varied from 8.2 to 20.3 mg gallic acid equivalents/g dry weight. DPPH radical scavenging effect of extracts decreased in the order of EF > BF > CE > PF > WF, with IC50 values ranging from 74.7 to 680.8 μg/mL. 2,2-azo-bis(3-Ethylbenzothiazoline-6-sulfoic acid) diammonium salt scavenging activity ranged from 0.118 to 0.236 mmol Trolox equivalence/mg extract. The extracts exhibited concentration-dependent reducing power, and EF showed the highest reducing ability. A satisfactory correlation (R2 > 0.826) between TPC and antioxidant activity was observed. In addition, EF, PF and CE exhibited potent anticancer effects on six cancer cell lines with IC50 values ranging from 40.1 to 166.3 μg/mL.Discussion and conclusion: The ethanol extract of A. oxyphylla fruit, especially the EF, was found to possess potent antioxidant and anticancer activities, and thus a great potential for the application in food and drug products.

The effect of photodynamic treatment on the morphological and mechanical properties of the HeLa cell line

High resolution imaging of biological structures and changes induced by various agents such as drugs and toxins is commonly performed by fluorescence and electron microscopy (EM). Although high-resolution imaging is possible with EM, the requirements for fixation and staining of samples for image contrast severely limits the study of living organisms. Atomic force microscopy (AFM), on the other hand, is capable of simultaneous nanometer spatial resolution and piconewton force detection, allowing detailed study of cell surface morphology and monitoring cytomechanical information. We present a method that images and studies mechanically characterized cells using AFM. We used a HeLa cell line (cervix carcinoma cell), which is sensitive to photodynamic treatment (PDT); growth media as a scanning surrounding; atomic force microscopy NT-MDT Aura for cytomechanical measurement; and scanning electron microscope Hitachi Su 6600 for control images of the cells. The modulus of elasticity for intact and photodynamically damaged cells can indicate mechanical changes to the main properties of cells. Cell elasticity changes can provide information on the degree or value of cell damage, for example after PDT. Measurements were carried out on approximately sixty cells, including three independent experiments on a control group and on sixty cells in a photodamaged group. Cells before PDT show higher elasticity: the median of Young´s modulus on the nucleus was 35.283 kPa and outside of the nucleus 107.442 kPa. After PDT, the median of Young's modulus on the nucleus was 61.144 kPa and outside of the nucleus was 193.605 kPa.

Condurango-glycoside-A fraction of Gonolobus condurango induces DNA damage associated senescence and apoptosis via ROS-dependent p53 signalling pathway in HeLa cells

Gonolobus condurango plant extract is used as an anticancer drug in some traditional systems of medicine including homeopathy, but it apparently lacks any scientific validation. Further, no detailed study is available to suggest whether condurango-glycoside-A (CGA), a major ingredient of condurango serves as a potent anticancer compound. Therefore, we investigated apoptosis-inducing ability of CGA against cervix carcinoma cells (HeLa). β-galactosidase-activity and DNA damage were critically studied at different time points; while induced DNA-damage was observed at 9-12th hours, senescence of cells appeared at a later stage (18th hour after CGA treatment), implicating thereby a possible role of DNA damage in inducing pre-mature cell senescence. Concurrently, the number of cells undergoing apoptosis increased along with increase in reactive oxygen species (ROS) generation. Expression of p53 was also up-regulated, indicating that apoptosis could have been mediated through p53 pathway. DCHFDA (4',6-Diamidino-2-phenylindole dihydrochloride) assay, acridine orange/ethidium bromide staining and annexin V/PI assay results collectively confirmed that apoptosis was induced by increased ROS generation. Reduction in proliferation of cells was further evidenced by the cell cycle arrest at G0/G1 stage. Expression profiles of certain relevant genes and proteins like p53, Akt, Bcl-2, Bax, cytochrome c and caspase 3 also provided evidence of ROS mediated p53 up-regulation and further boost in Bax expression and followed by cytochrome c release and activation of caspase 3. Overall results suggest that CGA initiates ROS generation, promoting up-regulation of p53 expression, thus resulting in apoptosis and pre-mature senescence associated with DNA damage.

Squamous cell carcinoma of cervix

Squamous cell carcinoma of cervix

Squamous cell carcinoma of cervix

Squamous cell carcinoma of cervix

Squamous cell carcinoma of cervix and breast cancer

Squamous cell carcinoma of cervix and breast cancer

Outcome analysis of locally advanced adenocarcinoma/adenosquamous carcinoma and squamous cell carcinoma of cervix after definitive radiotherapy.

e16503 Background: To compare tumor characteristics, treatment response, failure patterns, and clinical outcome of patients with cervix locally advanced adenocarcinoma (AC)/adenosquamous carcinoma (ASC) and squamous cell carcinoma (SCC). Methods: Medical records for 36 patients with stage IIB–IVA histologically proven cervix AC/ASC and 199 patients with SCC who received definitive radiotherapy (RT) or concurrent chemoradiotherapy (CCRT) between 1995 and 2009 were retrospectively reviewed. The clinicopathologic features, radiation dose, treatment response, recurrent pattern, metastases pattern, and survival outcome between histologic groups were evaluated. Results: Compared with the SCC subgroup of patients, patients with AC/ASC were significantly younger (mean age, 56 vs. 62 years, p=0.007), more of them without clinical symptoms were diagnosed by abnormal Papanicolaou smear findings (16.7% vs. 5.5%, p = 0.019), and less responded to treatment (complete response, 72.2% vs. 86.9%, p = 0.024). After a median follow-up of 59.3 months, patients with AC/ASC had worse 5-year recurrence-free survival (RFS) (33.7% vs. 58.7%, p = 0.046), worse 5-year distant metastasis-free survival (40.2% vs. 70.3%, p = 0.002), and trends toward worse 5-year local recurrence-free survival (62.4% vs. 75.7%, p = 0.127) and worse 5-year overall survival (OS) (40.1% vs. 58.0%, p = 0.062) than those with SCC. In univariate analysis, early FIGO stage and complete treatment response were found to be significantly associated with both RFS and OS. Histology of non-AC/ASC and Point A biologically equivalent doses in 2-Gy fractions >85Gy were significantly associated with better RFS, and CCRT was significantly associated with better OS. In multivariate analysis, complete treatment response remained significant factor for predicting better RFS, and early FIGO stage, CCRT and complete treatment response remained significant factors for predicting better OS. Conclusions: Cervix AC/ASC may behave more aggressively than does SCC. For this subgroup of cervical cancer, more comprehensively effective treatment is warranted.

Identification of head and neck squamous cell carcinoma biomarker candidates through proteomic analysis of cancer cell secretome

Protein biomarker discovery for early detection of head and neck squamous cell carcinoma (HNSCC) is a crucial unmet need to improve patient outcomes. Mass spectrometry-based proteomics has emerged as a promising tool for identification of biomarkers in different cancer types. Proteins secreted from cancer cells can serve as potential biomarkers for early diagnosis. In the current study, we have used isobaric tag for relative and absolute quantitation (iTRAQ) labeling methodology coupled with high resolution mass spectrometry to identify and quantitate secreted proteins from a panel of head and neck carcinoma cell lines. In all, we identified 2,472 proteins, of which 225 proteins were secreted at higher or lower abundance in HNSCC-derived cell lines. Of these, 148 were present in higher abundance and 77 were present in lower abundance in the cancer-cell derived secretome. We detected a higher abundance of some previously known markers for HNSCC including insulin like growth factor binding protein 3, IGFBP3 (11-fold) and opioid growth factor receptor, OGFR (10-fold) demonstrating the validity of our approach. We also identified several novel secreted proteins in HNSCC including olfactomedin-4, OLFM4 (12-fold) and hepatocyte growth factor activator, HGFA (5-fold). IHC-based validation was conducted in HNSCC using tissue microarrays which revealed overexpression of IGFBP3 and OLFM4 in 70% and 75% of the tested cases, respectively. Our study illustrates quantitative proteomics of secretome as a robust approach for identification of potential HNSCC biomarkers. This article is part of a Special Issue entitled: An Updated Secretome.

In Vitro Evaluation of Zn–Norfloxacin Complex as a Potent Cytotoxic and Antibacterial Agent, Proposed Model for DNA Binding

A tetrahedral Zn(II) complex with the second generation fluoroquinolone, norfloxacin, was prepared and characterized (NOR-Zn complex, NZC). The antibacterial efficiency of the NZC was tested on two Gram-positive and four Gram-negative bacteria by minimum inhibitory concentration method. The cytotoxic potential of NZC on MDA (human breast adenocarcinoma), Caco-2 (human colon adenocarcinoma), and Hela (human cervix carcinoma) cell lines was studied. The DNA interaction property of the NZC has been investigated using UV-vis, fluorescence, Fourier transform infrared, as well as cyclic voltammetry methods. Intrinsic binding constant (K(b)), thermodynamic, and other spectroscopic and voltammetric data indicate that the NZC has more affinity for DNA than for norfloxacin and interacted with DNA via two modes: electrostatic and outside hydrogen binding. The proposed DNA binding mode supports the large enhancement in the cytotoxicity and antibacterial activity of NZC.

Abstract 2882: Growth promoting effects of the Toll-like receptor 3 in Head and Neck carcinoma cells .

Abstract We have previously reported an intense expression of the Toll-Like receptor 3 (TLR3) in malignant cells from EBV-associated nasopharyngeal carcinomas and other Head and Neck carcinomas (Vérillaud et al., Infectious Agents and Cancer, in press). TLR3 is involved in innate immune response triggered by the binding of double-stranded RNA (dsRNA) of viral origin. Its consistent expression by malignant epithelial cells suggests that it has some functions in cell proliferation and survival. The aim of this work was to investigate these functions using 3 cell lines derived from Head and Neck squamous cell carcinomas : CNE1 (EBV-negative nasopharyngeal carcinoma), SQ20B (laryngeal carcinoma) and Fadu (hypopharyngeal carcinoma). Cell clones conditionally knocked-down for TLR-3 were derived from each parental cell line by stable transfection of a vector encoding a microRNA targeting TLR3 transcripts and whose expression is regulated by tetracycline. Stimulation of TLR3 in parental cells was done using small concentrations (250 ng/ml) of the synthetic TLR3-ligand poly(A/U). Modest but consistent growth enhancing effects were observed in deficient culture conditions marked by low concentrations of foetal calf serum and/or low amounts of nutrients and/or hypoxia. The influence of TLR3 on growth characteristics was investigated in more details using the CNE1 cells, both parental and conditionally knocked-down for TLR3. We found that under low-serum culture conditions, cell doubling time was shortened when TLR3 was stimulated by poly(A:U). On the opposite, knocking-down TLR3 resulted in a slower growth. To better understand the mechanism underlying the growth promoting effects of TLR3 in CNE1 cells, we assessed the metabolic changes induced by poly(A/U) stimulation or TLR3 silencing . Using the Seahorse Bioscience XF Glycolysis Stress Test Kit ® we found that the glycolytic capacity of CNE1 was significantly increased under stimulation by poly(A:U), whereas it was decreased under silencing of TLR3. Finally, we investigated the impact of TLR3-stimulation on the expression of HIF1-alpha which is a key transcription factor for cell adaptation to hypoxia and a major regulator of glucid metabolism. Western blot analysis showed that HIF1-alpha could be detected even in normoxic conditions when CNE1 cells were treated by poly(A:U) for 24 hours in low serum conditions. On the other hand, knocking-down TLR3 in CNE1 cells resulted in a decrease in the cellular concentration of HIF1-alpha under hypoxia. Overall, these results suggest that TLR3-stimulation induces metabolic changes favorable to cell growth in deficient culture conditions. Future investigations will intend to investigate: 1) the in vivo functions of TLR-3 using xenografted CNE1 cells; 2) the contribution of HIF1-alpha to the metabolic changes resulting from TLR3 stimulation; 3) the nature and origin of the endogenous ligands of TLR3. Citation Format: Benjamin Vérillaud, Mélanie Gressette, Philippe Herman, Anne-Sophie Jimenez, Pierre Busson. Growth promoting effects of the Toll-like receptor 3 in Head and Neck carcinoma cells . [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 2882. doi:10.1158/1538-7445.AM2013-2882

Abstract 479: ATM pathway upregulation of combination therapy with radiation and MSC-based IL-12M gene vaccine, Gx-051, in allograft animal model.

Abstract PURPOSE: In spite of the powerful therapeutic effects of radiotherapy, the attempts to combined radiotherapy with others have so far kept going on to find better therapeutic protocol for patients in the head and neck cancer. Previously, we announced some part of anticancer effects of combined radiotherapy with Gx-051 named as mesenchymal stem cell (MSC) expressing modified interleukin-12 (MSCs/IL-12M). It is a novel component of human immune system to attack cancer cells by the recruitment of immune cells with a high specificity. Thus, in the present study, we have tried to reveal another therapeutic effect of combined radiation and immunotherapy, compared to the treatment with each single modality in allograft animal model. METHODS AND MATERIALS: AT-84 cells, one of murine head and neck carcinoma cells, were injected for allograft induction on C3H/HeN mice. All the mice were randomly divided into 4 groups; control (non-treated), radiation only-treated, Gx-051 only-treated, and irradiation with Gx-051-treated. Intra-tumoral injection of Gx-051 (adopted within 24hr after irradiation) as well as 8 Gy-local irradiation was carried out, totally 4 times (twice a week for 2 weeks). To clarify the anti-tumoral effect of radiation with Gx-051 administration, we provided reasonable evidences; tumor volume, levels of IL-12 and IFN-r, and the changes in signaling pathway related-protein expression level. RESULTS: In the present study, we could confirm that combined treatment with radiation and Gx-051 significantly enhanced the anti-tumor effect. The proliferation of cancer cell was markedly inhibited in all the treated groups compared to non-treated group (p<0.05) as down-regulating of PCNA expression. Also, via mediating the expression of ATM and CDK2, the combined treatment group (radiotherapy with Gx-051) significantly enhanced the activation of ATM pathway-related proteins (p<0.05). The tumor suppress gene, p53, was highly expressed and the one of typical oncogenes, Met, was suppressed in combined treatment group compared to others. In addition, expression of p38-MAPk as one of the cell stress markers was decreased in combined treatment group compared to others. CONCLUSIONS: We determined the combined radiotherapy with Gx-051 improved the responses to HNSCC by AT-84 via the activation of ATM pathway that influenced cell proliferation, apoptosis, and regulation of tumor suppress gene and oncogene. These results gave us the partial rationale to the further study about the combined therapy of radiation with Gx-051 for the advanced treatment of human HNSCC. This study was supported by grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare (A091205) and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2012-0004140). Citation Format: Jiyoung Yeo, Eun-Ji Park, Kwang-Jae Cho, Kyung Min Kim, Young-chul Sung, Jun-Ook Park, In-chul Nam, Chung-su Kim, Min-Sik Kim. ATM pathway upregulation of combination therapy with radiation and MSC-based IL-12M gene vaccine, Gx-051, in allograft animal model. [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 479. doi:10.1158/1538-7445.AM2013-479

C2-Streptavidin Mediates the Delivery of Biotin-Conjugated Tumor Suppressor Protein P53 into Tumor Cells

We have previously generated a recombinant C2-streptavidin fusion protein for the delivery of biotin-labeled molecules of low molecular weight into the cytosol of mammalian cells. A nontoxic moiety of Clostridium botulinum C2 toxin mediates the cellular uptake, whereas the streptavidin unit serves as a binding platform for biotin-labeled cargo molecules. In the present study, we used the C2-streptavidin transporter to introduce biotin-conjugated p53 protein into various mammalian cell lines. The p53 tumor suppressor protein is inactivated in many human cancers by multiple mechanisms and therefore the restoration of its activity in tumor cells is of great therapeutic interest. Recombinant p53 was expressed in insect cells and biotin-labeled. Biotin-p53 retained its specific high-affinity DNA-binding as revealed by gel-shift analysis. Successful conjugation of biotin-p53 to the C2-streptavidin transporter was monitored by an overlay blot technique and confirmed by real-time surface plasmon resonance, providing a KD-value in the low nM range. C2-streptavidin significantly enhanced the uptake of biotin-p53 into African Green Monkey (Vero) epithelial cells as shown by flow cytometry. Using cell fractionation, the cytosolic translocation of biotin-p53 was detected in Vero cells as well as in HeLa cervix carcinoma cells. In line with this finding, confocal microscopy displayed cytoplasmic staining of biotin-p53 in HeLa and HL60 leukemia cells. Internalized biotin-p53 partially colocalized with early endosomes, as confirmed by confocal microscopy. In conclusion, our results demonstrate the successful conjugation of biotin-p53 to C2-streptavidin and its subsequent receptor-mediated endocytosis into different human tumor cell lines.

Stimuli-Responsive Magnetic Nanomicelles as Multifunctional Heat and Cargo Delivery Vehicles

Hybrid nanoarchitectures are among the most promising nanotechnology-enabled materials for biomedical applications. Interfacing of nanoparticles with active materials gives rise to the structures with unique multiple functionality. Superparamagnetic iron oxide nanoparticles particles SPION are widely employed in the biology and in developing of advanced medical technologies. Polymeric micelles offer the advantage of multifunctional carriers which can serve as delivery vehicles carrying nanoparticles, hydrophobic chemotherapeutics and other functional materials and molecules. Stimuli-responsive polymers are especially attractive since their properties can be modulated in a controlled manner. Here we report on multifunctional thermo-responsive poly(N-isopropylacrylamide-co-acrylamide)-block-poly(ε-caprolactone) random block copolymer micelles as magnetic hyperthermia-mediated payload release and imaging agents. The combination of copolymers, nanoparticles and doxorubicin drug was tailored the way that the loaded micelles were cable to respond to magnetic heating at physiologically-relevant temperatures. A surface functionalization of the micelles with the integrin β4 antibody and consequent interfacing of the resulting nanobio hybrid with squamous head and neck carcinoma cells which is known to specifically over-express the A9 antigen resulted in concentration of the micelles on the surface of cells. No inherent cytotoxicity was detected for the magnetic micelles without external stimuli application. Furthermore, SPION-loaded micelles demonstrate significant MRI contrast enhancement abilities.