Giselle V. Ripoll

The Functional Interaction between Acyl-CoA Synthetase 4, 5-Lipooxygenase and Cyclooxygenase-2 Controls Tumor Growth: A Novel Therapeutic Target

The acyl-CoA synthetase 4 (ACSL4), which esterify mainly arachidonic acid (AA) into acyl-CoA, is increased in breast, colon and hepatocellular carcinoma. The transfection of MCF-7 cells with ACSL4 cDNA transforms the cells into a highly aggressive phenotype and controls both lipooxygenase-5 (LOX-5) and cyclooxygenase-2 (COX-2) metabolism of AA, suggesting a causal role of ACSL4 in tumorigenesis. We hypothesized that ACSL4, LOX-5 and COX-2 may constitute potential therapeutic targets for the control of tumor growth. Therefore, the aim of this study was to use a tetracycline Tet-Off system of MCF-7 xenograft model of breast cancer to confirm the effect of ACSL4 overexpression on tumor growth in vivo. We also aim to determine whether a combinatorial inhibition of the ACSL4-LOX-COX-2 pathway affects tumor growth in vivo using a xenograft model based on MDA-MB-231 cells, a highly aggressive breast cancer cell line naturally overexpressing ACSL4. The first novel finding is that stable transfection of MCF-7 cells with ACSL4 using the tetracycline Tet-Off system of MCF-7 cells resulted in development of growing tumors when injected into nude mice. Tumor xenograft development measured in animals that received doxycycline resulted in tumor growth inhibition. The tumors presented marked nuclear polymorphism, high mitotic index and low expression of estrogen and progesterone receptor. These results demonstrate the transformational capacity of ACSL4 overexpression. We examined the effect of a combination of inhibitors of ACSL4, LOX-5 and COX-2 on MDA-MB-231 tumor xenografts. This treatment markedly reduced tumor growth in doses of these inhibitors that were otherwise ineffective when used alone, indicating a synergistic effect of the compounds. Our results suggest that these enzymes interact functionally and form an integrated system that operates in a concerted manner to regulate tumor growth and consequently may be potential therapeutic targets for the control of proliferation as well as metastatic potential of cancer cells.

Complete Antitumor Protection by Perioperative Immunization with GM3/VSSP Vaccine in a Preclinical Mouse Melanoma Model

Purpose: The GM3/VSSP vaccine is composed of very small sized proteoliposomes resulting from the hydrophobic conjugation of GM3 ganglioside with membrane proteins from Neisseria meningitidis. Previously, we showed that preventive vaccination with GM3/VSSP induces a specific antitumor response and elicits the rejection of syngeneic GM3-positive melanoma cells in immunized mice. Our aim was to explore the antitumor properties of perioperative GM3/VSSP vaccination in a preclinical mouse model. Experimental Design: The highly metastatic B16F10 mouse melanoma was used to investigate perioperative vaccination with GM3/VSSP. The vaccine was administered i.m. in doses of 120 μg emulsified with the adjuvant Montanide ISA 51 at weekly or biweekly intervals, and s.c. tumors were excised 25 to 31 days after tumor cell implantation. The persistence of antitumor protection and dose dependency was also examined in preimmunized animals. To evaluate the immune performance of tumor-bearing and tumor-operated mice, ovoalbumin-specific delayed-type hypersensitivity, cytokine secretion, and cell proliferation responses were studied. Results: Surgical excision of B16F10 tumors improved survival, and perioperative immunization with four biweekly GM3/VSSP doses yielded survival for all animals (P = 0.04; log-rank test). Mice showed neither local recurrence nor lung metastasis at the end of the experiment. An impairment of CD4+ T-cell responses was observed in tumor-bearing animals measured as neoantigen-specific delayed-type hypersensitivity, with a significant recovery after surgery. A strong interleukin-4 secretion was induced in B16F10-operated mice, whereas IFN-γ remained unaffected. Conclusion: Preclinical evidence suggests that GM3/VSSP vaccine might have therapeutic potential to induce antitumor immunity in patients with minimal residual disease after surgery, thereby preventing or prolonging the time to recurrence.

Antiproliferative effect of 1-deamino-8-D-arginine vasopressin analogs on human breast cancer cells

BACKGROUND Desmopressin (dDAVP), a synthetic nonapeptide derivative of arginine vasopressin, is a safe antidiuretic and hemostatic compound that acts as a selective agonist for the vasopressin V2 membrane receptor (V2R). It is known that dDAVP can inhibit progression of residual metastatic cells in preclinical models. Among other mechanisms, the compound induces an agonist effect on V2R present in tumor cells. RESULTS/DISCUSSION Looking for novel analogs with improved anti-tumor activity, positions 4 and 5, at the conformational peptide loop, were substituted. The analog [V(4)Q(5)]dDAVP ([4-valine 5-glutamine] desmopressin) exhibited a significantly higher antiproliferative effect than dDAVP in cultures of MCF-7, a V2R-expressing human breast carcinoma cell line. The chiral isomer of this analog and tetrapeptide fragments corresponding to the loop region were also assessed. CONCLUSION Preclinical evaluation of the anti-tumor activity of [V(4)Q(5)]dDAVP in animal models is warranted.

Structure‑activity relationship of 1‑desamino‑8‑D‑arginine vasopressin as an antiproliferative agent on human vasopressin V2 receptor‑expressing cancer cells

The synthetic nonapeptide 1‑desamino‑8‑D‑arginine vasopressin (dDAVP) can reduce tumor cell growth through agonist action on the vasopressin V2 receptor. A structure‑antiproliferative activity relationship analysis of dDAVP was performed using the alanine scanning technique on the aggressive MDA‑MB‑231 human breast carcinoma cell line. The results from this analysis demonstrated that the amino acids located at the loop of dDAVP are important for the antiproliferative activity of dDAVP, highlighting the key role of the N‑terminal region of the peptide in the interaction with the tumor cell surface receptor. The findings from this study present novel strategies for designing improved compounds with enhanced stability for cancer therapy.

Peptide Agonists of Vasopressin V2 Receptor Reduce Expression of Neuroendocrine Markers and Tumor Growth in Human Lung and Prostate Tumor Cells

Neuroendocrine tumors (NETs) comprise a heterogeneous group of malignancies that express neuropeptides as synaptophysin, chromogranin A (CgA), and specific neuronal enolase (NSE), among others. Vasopressin (AVP) is a neuropeptide with an endocrine, paracrine, and autocrine effect in normal and pathological tissues. AVP receptors are present in human lung, breast, pancreatic, colorectal, and gastrointestinal tumors. While AVP V1 receptors are associated with stimulation of cellular proliferation, AVP V2 receptor (V2r) is related to antiproliferative effects. Desmopressin (dDAVP) is a synthetic analog of AVP that acts as a selective agonist for the V2r, which shows antitumor properties in breast and colorectal cancer models. Recently, we developed a derivative of dDAVP named [V4Q5]dDAVP, which presents higher antitumor effects in a breast cancer model compared to the parental compound. The goal of present work was to explore the antitumor properties of the V2r agonist dDAVP and its novel analog [V4Q5]dDAVP on aggressive human lung (NCI-H82) and prostate cancer (PC-3) cell lines with neuroendocrine (NE) characteristics. We study the presence of specific NE markers (CgA and NSE) and V2r expression in NCI-H82 and PC-3. Both cell lines express high levels of NE markers NSE and CgA but then incubation with dDAVP diminished expression levels of both markers. DDAVP and [V4Q5]dDAVP significantly reduced proliferation, doubling time, and migration in both tumor cell cultures. [V4Q5]dDAVP analog showed a higher cytostatic effect than dDAVP, on cellular proliferation in the NCI-H82 cell line. Silencing of V2r using small interfering RNA significantly attenuated the inhibitory effects of [V4Q5]dDAVP on NCI-H82 cell proliferation. We, preliminarily, explored the in vivo effect of dDAVP and [V4Q5]dDAVP on NCI-H82 small cell lung cancer xenografts. Treated tumors (0.3 μg kg−1, thrice a week) grew slower in comparison to vehicle-treated animals. In this work, we demonstrated that the specific agonists of V2r, dDAVP, and [V4Q5]dDAVP displays antitumor capacity on different human models of lung and prostate cancers with NE features, showing their potential therapeutic benefits in the treatment of these aggressive tumors.

Implication of von Willebrand Factor as a Regulator of Tumor Cell Metastasis: Potential Perioperative Use of Desmopressin and Novel Peptide Analogs

Fil: Ripoll, Giselle Vanina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnologia. Laboratorio de Oncologia Molecular; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas; Argentina

Search of vasopressin analogs with antiproliferative activity on small-cell lung cancer: drug design based on two different approaches

AIM Development of compounds with therapeutic application requires the interaction of different disciplines. Several tumors express vasopressin (AVP; arginine vasopressin) receptors with contrasting effects depending on receptor subtype. Desmopressin (dDAVP) is an AVP-selective analog with antiproliferative properties. In this work, an evolutionary approach and a rational strategy were applied in order to design novel AVP analogs. RESULTS We designed two novel analogs; dDInotocin (dDINT, insect analog), and [V4Q5]dDAVP, and demonstrated the importance of the dDAVP conformational loop for its antiproliferative activity. [V4Q5] dDAVP showed major cytostatic effect on lung cancer cells than dDAVP and its cytostatic effect was abolished by V2R blockade. CONCLUSION Combination of these strategies could provide the basis for future studies for the development of improved compounds with potential therapeutic applications.

Preclinical Efficacy of [V 4 Q 5 ]dDAVP, a Second Generation Vasopressin Analog, on Metastatic Spread and Tumor-Associated Angiogenesis in Colorectal Cancer

Purpose Control of metastatic spread of colorectal cancer (CRC) remains as a major therapeutic challenge. [V4 Q5 ]dDAVP is a vasopressin peptide analog with previously reported anticancer activity against carcinoma tumors. By acting as a selective agonist of arginine vasopressin type 2 membrane receptor (AVPR2) present in endothelial and tumor cells, [V4Q5]dDAVP is able to impair tumor aggressiveness and distant spread. Our aim was to evaluate the potential therapeutic benefits of [V4Q5]dDAVP on highly aggressive CRC disease using experimental models with translational relevance. Materials and Methods Murine CT-26 and human Colo-205 AVPR2-expressing CRC cell lines were used to test the preclinical efficacy of [V4Q5]dDAVP, both in vitro and in vivo. Results In syngeneic mice surgically implanted with CT-26 cells in the spleen, sustained intravenous treatment with [V4Q5]dDAVP (0.3 µg/kg) dramatically impaired metastatic progression to liver without overt signs of toxicity, and also reduced experimental lung colonization. The compound inhibited in vivo angiogenesis driven by Colo-205 cells in athymic mice, as well as in vitro endothelial cell migration and capillary tube formation. [V4Q5]dDAVP exerted AVPR2-dependent cytostatic activity in vitro (IC50 1.08 µM) and addition to 5-fluorouracil resulted in synergistic antiproliferative effects both in CT-26 and Colo-205 cells. Conclusion The present preclinical study establishes for the first time the efficacy of [V4Q5]dDAVP on CRC. These encouraging results suggest that the novel second generation vasopressin analog could be used for the management of aggressive CRC as an adjuvant agent during surgery or to complement standard chemotherapy, limiting tumor angiogenesis and metastasis and thus protecting the patient from CRC recurrence.

Abstract 2321: The novel vasopressin analog [4-valine 5-glutamine] desmopressin inhibits tumor growth and angiogenesis in V2 receptor-expressing breast cancer xenografts

Desmopressin (dDAVP), a synthetic nonapeptide derivative of arginine vasopressin, is a safe antidiuretic and hemostatic compound that acts as a selective agonist for the vasopressin V2 membrane receptor (V2R). This receptor is expressed in kidney collecting tubes, endothelium and also in some tumor cells. We previously reported that dDAVP can inhibit progression of residual metastatic cells in preclinical models. Among other mechanisms, the compound induces an agonist effect on V2R present in tumor cells. This cyclic nonapeptide was substituted in positions 4 and 5 searching for compounds with improved biological activity (Iannucci et al., Fut. Med. Chem. 2011). Such positions belong to the conformational peptide loop which has a key role in ligand-receptor interaction. In this study, we evaluated the effects of the novel analog [4-valine 5-glutamine] dDAVP ([V4Q5]dDAVP) on cell proliferation and migration in vitro, and tumor growth and angiogenesis in vivo using the MDA-MB-231 triple-negative V2R-expressing human breast cancer cell line. We first examined peptide ability to modify in vitro cell growth of MDA-MB-231 cells by the MTT assay. Incubation with [V4Q5]dDAVP analog (100-1500 nM) during 72 h significantly reduced cell proliferation showing a better performance at high concentrations than the parental compound dDAVP. When studying the effect on cell cycle, flow cytometric analysis showed that a 24-h treatment with [V4Q5]dDAVP resulted in an arrest of MDA-MB-231 cells in G0/G1 phase. Next, we used wound migration and transwell chemotaxis assays to study the ability of [V4Q5]dDAVP to inhibit tumor cell motility. After a 16-h treatment, MDA-MB-231 migration and chemotaxis was impaired by 30-60%. To asses the in vivo effect of the compound on tumor growth, nu/nu mice where implanted s.c. with MDA-MB-231 cells. Three weekly i.v. doses of [V4Q5]dDAVP (0.3 µg/kg/dose) reduced by 40% the subcutaneous tumor volume compared with vehicle-treated controls after a 4- week treatment. Finally, to evaluate the in vivo angiostatic effect, a modified Matrigel plug assay was used. Intravenous administration of [V4Q5]dDAVP (0.3 µg/kg/dose thrice a week) significantly reduced by 18% MDA-MB-231 cell-induced angiogenesis as measured by hemoglobin content in plugs recovered from mice 14 days after tumor cell inoculation. These in vivo effects are probably due to a dual action of [V4Q5]dDAVP involving angiogenesis inhibition mechanisms and direct cytostatic action. In conclusion, this study indicates that the novel peptide compound [V4Q5]dDAVP exerts a remarkable and improved antitumor and antiangiogenic effect in comparison to parental peptide dDAVP. Increased hidrophobicity at the conformational peptide loop by replacing Gln for Val may play a key role on ligand-receptor interaction, thus enhancing agonist effect on V2R present in tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2321. doi:1538-7445.AM2012-2321

Abstract 5473: Antiproliferative and antimetastasic activity of a novel vasopressin V2 receptor peptide agonist in a breast cancer model

Desmopressin (1-deamino-8-D-arginine vasopressin) is a synthetic analog of vasopressin with antimetastatic properties. The molecule is a well known and safe hemostatic compound that acts as a selective agonist for the vasopressin V2 membrane receptor, which is expressed in endothelium and also in some tumor cells. Previous data revealed that perioperative administration of desmopressin can minimize the spread and survival of residual cancer cells. This cyclic nonapeptide was substituted in positions 4 and 5 searching for compounds with improved biological activity and half life. Such positions belong to the disulphide loop (between positions 1-6), which have a major interaction with its receptor. In this work, we evaluated the in vitro biological activity of a novel analog designated VQ (1-deamino-4-valine-5-glutamine-8-D-arginine vasopressin) in MCF7 human mammary carcinoma cells, as well as its in vivo antitumor action in combination with chemotherapy using the syngeneic F3II mammary carcinoma model in Balb/c mice. Because V2 receptor signaling has been associated with cytostatic effects, we have examined peptide ability to modify in vitro cell proliferation of MCF7 cells expressing V2 receptor. Incubation with VQ analog (100-1500 nM) during 72 h significantly reduced cell proliferation (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5473.