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Masuelli L.,University of Rome La Sapienza | Marzocchella L.,University of Rome Tor Vergata | Focaccetti C.,University of Rome Tor Vergata | Lista F.,Centro Studi e Ricerche Sanita e Veterinaria Esercito | And 9 more authors.
Cancer Immunology, Immunotherapy | Year: 2010

Recombinant vaccinia virus has been widely employed as a cancer vaccine in several clinical trials. In this study we explored, employing BALB/c mice transgenic for the rat neu oncogene, the ability of the recombinant vaccinia virus neu (rV-neuT) vaccine to inhibit growth of neu+ mammary carcinomas and whether the efficacy of vaccination was dependent on: (a) carcinogenesis stage at which the vaccination was initiated; (b) number of vaccinations and (c) route of delivery (systemic vs. local). BALB-neuT mice were vaccinated one, two and three times by subcutaneous (s.c.) and intramammary gland (im.g.) injection with rV-neuT or V-wt (wild-type vaccinia virus) starting at the stage in which mouse mammary gland displays atypical hyperplasia, carcinoma in situ or invasive carcinoma. We demonstrated that vaccination using rV-neuT was more effective when started at an earlier stage of mammary carcinogenesis and after three vaccinations. The im.g. vaccination was more effective than the s.c. vaccination in inhibiting mammary carcinogenesis, eliciting anti-Neu antibodies, increasing anti-Neu IgG2a/G3 isotypes and inducing antibodies able to trigger mammary tumor cells apoptosis and antibody-dependent cellular cytotoxicity. The better protective ability of rV-neuT im.g. vaccination was associated with its capacity to induce a superior degree of in vivo mammary cancer cells apoptosis. Our research suggests that intratumoral vaccination using recombinant vaccinia virus could be employed to increase the activity of a genetic cancer vaccine. This study may have important implications for the design of cancer vaccine protocols for the treatment of breast cancer and of accessible tumors using recombinant vaccinia virus. © 2010 Springer-Verlag.


Masuelli L.,University of Rome La Sapienza | Fantini M.,University of Rome Tor Vergata | Benvenuto M.,University of Rome Tor Vergata | Sacchetti P.,University of Rome La Sapienza | And 9 more authors.
Journal of Translational Medicine | Year: 2014

Background: The antitumor activity induced by intratumoral vaccination with poxvirus expressing a tumor antigen was shown to be superior to that induced by subcutaneous vaccination. Salivary gland carcinomas overexpress ErbB2. Trastuzumab, a monoclonal antibody to ErbB2, was proposed for salivary gland tumors treatment. We explored the effectiveness of intratumoral vaccination with the recombinant vaccinia virus ErbB2/Neu (rV-neuT) vaccine in hampering the growth of transplanted Neu-overexpressing BALB-neuT salivary gland cancer cells (SALTO) in BALB-neuT mice.Methods: BALB-neuT male mice were subcutaneously injected with SALTO tumor cells and intratumorally vaccinated twice with different doses of either rV-neuT or V-wt (wild-type). Tumors were measured weekly. The presence of anti-ErbB2/Neu antibodies was assayed by ELISA, immunoprecipitation or indirect immunofluorescence. Biological activity of immune sera was investigated by analyzing antibody-dependent cellular cytotoxicity (ADCC), SALTO cells proliferation and apoptosis, ErbB2/Neu receptor down regulation and ERK1/2 phosphorylation. Anti-Neu T cell immunity was investigated by determining the release of IL-2 and IFN-gamma in T cells supernatant. Survival curves were determined using the Kaplan-Meier method and compared using the log-rank test. Differences in tumor volumes, number of apoptotic cells, titer of the serum, percentage of ADCC were evaluated through a two-tailed Student's t-test.Results: rV-neuT intratumoral vaccination was able to inhibit the growth of SALTO cancer cells in a dose-dependent manner. The anti-Neu serum titer paralleled in vivo antitumor activity of rV-neuT vaccinated mice. rV-neuT immune serum was able to mediate ADCC, inhibition of SALTO cells proliferation, down regulation of the ErbB2/Neu receptor, inhibition of ERK1/2 phosphorylation and induction of apoptosis, thus suggesting potential mechanisms of in vivo tumor growth interference. In addition, spleen T cells of rV-neuT vaccinated mice released IFN-gamma and IL-2 upon in vitro stimulation with several Neu-specific peptides located in the extracellular domain of Neu sequence.Conclusions: rV-neuT intratumoral vaccination could be employed to induce an efficient antitumor response and reject transplanted salivary gland tumors. Our findings may have important implications for the design of cancer vaccine protocols for the treatment of salivary gland tumors and other accessible tumors using intratumoral injection of recombinant vaccinia virus. © 2014 Masuelli et al.; licensee BioMed Central Ltd.


PubMed | University of Rome La Sapienza, University of Rome Tor Vergata, Centro Studi e Ricerche Sanita e Veterinaria Esercito and Foro Italico University of Rome
Type: Journal Article | Journal: Oncotarget | Year: 2014

The survival rate of head and neck squamous cell carcinomas (HNSCC) patients has not considerably changed over the last two decades. Polyphenols inhibit the growth of cancer cells. We determined whether the combination of Resveratrol (RES) and Curcumin (CUR) enhanced their in vitro and in vivo antitumor activities on HNSCC cell lines compared to the single compounds. We provide evidence that RES potentiated the apoptotic effect and reduced the IC50 of CUR on HNSCC cell lines. The model of compounds interaction indicated the onset of an additive effect of the two compounds compared to the single treatment after decrease of their concentrations. RES+CUR compared to CUR increased the PARP-1 cleavage, the Bax/Bcl-2 ratio, the inhibition of ERK1 and ERK2 phosphorylation, and the expression of LC3 II simultaneously with the formation of autophagic vacuoles. RES and CUR induced cytoplasmic NF-B accumulation. RES+CUR administrations were safe in BALB/c mice and reduced the growth of transplanted salivary gland cancer cells (SALTO) more efficiently than CUR. Overall, combinations of CUR and RES was more effective in inhibiting in vivo and in vitro cancer growth than the treatment with CUR. Additional studies will be needed to define the therapeutic potential of these compounds in combination.


Benvenuto M.,University of Rome Tor Vergata | Sileri P.,University of Rome Tor Vergata | Rossi P.,University of Rome Tor Vergata | Masuelli L.,University of Rome La Sapienza | And 13 more authors.
Journal of Translational Medicine | Year: 2015

Background: Tumor associated antigens are useful in colorectal cancer (CRC) management. The ribosomal P proteins (P0, P1, P2) play an important role in protein synthesis and tumor formation. The immunogenicity of the ribosomal P0 protein in head and neck, in breast and prostate cancer patients and the overexpression of the carboxyl-terminal P0 epitope (C-22 P0) in some tumors were reported. Methods: Sera from 72 colorectal tumor patients (67 malignant and 5 benign tumors) were compared with 73 healthy donor sera for the presence of antibodies to CEA, EGFR, ErbB2 and ribosomal P proteins by western blotting or ELISA. Expression of the C-22 P0 epitope on tissues and colon cancer cells was determined by immunoperoxidase staining and indirect immunofluorescence/western blotting, respectively, employing MAb 2B2. Biological effects of MAb 2B2 on colon cancer cells were assessed by the Sulforhodamine B cell proliferation assay, trypan blue exclusion test and cleaved caspase-3 detection. Fisher's exact test was used to compare the number of auto-antibodies positive patients with healthy donors. Variation in the C-22 P0 expression, and in the number of apoptotic cells was evaluated by Student's t-test. Variation in cell survival and cell death was evaluated by Newman-Keuls test. Results: No significant humoral response was observed to CEA, EGFR and ErbB2 in CRC patients. Conversely, 7 out of 67 CRC patient sera reacted to ribosomal P proteins. The prevalence of P proteins auto-antibodies in CRC patients was significant. Five patients showed restricted P0 immunoreactivity, while two patients reacted simultaneously to all P proteins. The C-22 P0 epitope was homogenously expressed both in malignant tumors and the adjacent mucosa, but the intensity of expression was higher in the tumor. Starved colon cancer cells showed a higher C-22 P0 epitope plasma membrane expression compared to control cells. MAb 2B2 inhibited colon cancer cell growth and induced cell death in a dose dependent manner. Conclusions: Our study shows a spontaneous humoral immune response to ribosomal P0 protein in CRC patients and the inhibition of in vitro cancer cell growth after C-22 P0 epitope targeting. The ribosomal P0 protein might be a useful immunological target in CRC patients. © 2015 Benvenuto et al.; licensee BioMed Central.


PubMed | University of Rome La Sapienza, University of Rome Tor Vergata, CNR Institute of Neuroscience and Centro Studi e Ricerche Sanita e Veterinaria Esercito
Type: | Journal: Journal of translational medicine | Year: 2015

Tumor associated antigens are useful in colorectal cancer (CRC) management. The ribosomal P proteins (P0, P1, P2) play an important role in protein synthesis and tumor formation. The immunogenicity of the ribosomal P0 protein in head and neck, in breast and prostate cancer patients and the overexpression of the carboxyl-terminal P0 epitope (C-22 P0) in some tumors were reported.Sera from 72 colorectal tumor patients (67 malignant and 5 benign tumors) were compared with 73 healthy donor sera for the presence of antibodies to CEA, EGFR, ErbB2 and ribosomal P proteins by western blotting or ELISA. Expression of the C-22 P0 epitope on tissues and colon cancer cells was determined by immunoperoxidase staining and indirect immunofluorescence/western blotting, respectively, employing MAb 2B2. Biological effects of MAb 2B2 on colon cancer cells were assessed by the Sulforhodamine B cell proliferation assay, trypan blue exclusion test and cleaved caspase-3 detection. Fishers exact test was used to compare the number of auto-antibodies positive patients with healthy donors. Variation in the C-22 P0 expression, and in the number of apoptotic cells was evaluated by Students t-test. Variation in cell survival and cell death was evaluated by Newman-Keuls test.No significant humoral response was observed to CEA, EGFR and ErbB2 in CRC patients. Conversely, 7 out of 67 CRC patient sera reacted to ribosomal P proteins. The prevalence of P proteins auto-antibodies in CRC patients was significant. Five patients showed restricted P0 immunoreactivity, while two patients reacted simultaneously to all P proteins. The C-22 P0 epitope was homogenously expressed both in malignant tumors and the adjacent mucosa, but the intensity of expression was higher in the tumor. Starved colon cancer cells showed a higher C-22 P0 epitope plasma membrane expression compared to control cells. MAb 2B2 inhibited colon cancer cell growth and induced cell death in a dose dependent manner.Our study shows a spontaneous humoral immune response to ribosomal P0 protein in CRC patients and the inhibition of in vitro cancer cell growth after C-22 P0 epitope targeting. The ribosomal P0 protein might be a useful immunological target in CRC patients.

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