Hephzibah Rani S. Tagaram

Nanoliposomal ceramide prevents in vivo growth of hepatocellular carcinoma

Background and objectives Hepatocellular carcinoma (HCC) affects an increasing number of people worldwide. The poor survival rate of patients with HCC is manifested by an aggressive and metastatic phenotype, as well as a poor response to common therapeutic strategies. The purpose of this study was to evaluate the efficacy of nanoliposomal C6-ceramide as an antineoplastic agent in an in vivo model of human HCC. Methods The growth-arresting and pro-apoptotic properties of nanoliposomal C6-ceramide were first evaluated in vitro in human SK-HEP-1 cells by assessing cellular viability, caspase 3/7 activity, annexin-V expression, DNA fragmentation, cell cycle distribution and AKT phosphorylation. SK-HEP-1 cells were then engrafted subcutaneously into athymic nude mice and nanoliposomal C6-ceramide was administered by tail vein injection. Tumour size was monitored over time, followed by excision of tumours to evaluate tumour vascularisation, proliferation, apoptosis and cellular signalling. Results Nanoliposomal C6-ceramide, but not ghost (no ceramide) nanoliposomes, induced apoptotic cell death of SK-HEP-1 cells in vitro, concomitant with an accumulation of cells in the G2 phase of the cell cycle and decreased phosphorylation of AKT. Systemic administration of nanoliposomal C6-ceramide to mice engrafted with SK-HEP-1 tumours reduced tumour vascularisation and proliferation, induced tumour cell apoptosis, decreased phosphorylation of AKT and ultimately blocked tumour growth. Conclusions These studies show that nanoliposomal ceramide is an efficacious antineoplastic agent for the treatment of in vitro and in vivo models of human HCC.

Combinatorial therapies improve the therapeutic efficacy of nanoliposomal ceramide for pancreatic cancer

Poor prognosis cancers, such as pancreatic cancer, represent inherent challenges for ceramide-based nanotherapeutics due to metabolic pathways, which neutralize ceramide to less toxic or pro-oncogenic metabolites. We have recently developed a novel 80 nanometer diameter liposomal formulation that incorporates 30 molar percent C6-ceramide, a bioactive lipid that is pro-apoptotic to many cancer cells, but not to normal cells. In this manuscript, we evaluated the efficacy of combining nanoliposomal C6-ceramide (Lip-C6) with either gemcitabine or an inhibitor of glucosylceramide synthase. We first assessed the biological effect of Lip-C6 in PANC-1 cells, a gemcitabine-resistant human pancreatic cancer cell line, and found that low doses alone did not induce cell toxicity. However, cytotoxicity was achieved by combining Lip-C6 with either non-toxic sub-therapeutic concentrations of gemcitabine or with the glucosylceramide synthase inhibitor D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP). Furthermore, these combinations with Lip-C6 cooperatively inhibited PANC-1 tumor growth in vivo. Mechanistically, Lip-C6 inhibited pro-survival Akt and Erk signaling, whereas the nucleoside analog gemcitabine did not. Furthermore, by including PDMP within the nanoliposomes, which halted ceramide neutralization as evidenced by LC-MS3, the cytotoxic effects of Lip-C6 were enhanced. Collectively, we have demonstrated that nanoliposomal ceramide can be an effective anti-pancreatic cancer therapeutic in combination with gemcitabine or an inhibitor of ceramide neutralization.

Synthesis and biological evaluation of a novel class of isatin analogs as dual inhibitors of tubulin polymerization and Akt pathway

A novel series of 5,7-dibromoisatin analogs were synthesized and evaluated for their cytotoxicities against four human cancer cell lines including colon HT29, breast MCF-7, lung A549 and melanoma UACC903. Analogs 6, 11 and 13 displayed good in vitro anticancer activity on the HT29 human colon cancer cell line in the 1 μM range. Analogs 5, 9 and 12, containing a selenocyanate group in the alkyl chain were the most promising compounds on the breast cancer MCF-7 cell line. Biological assays relating to apoptosis were performed to understand the mechanism of action of these analogs. Compounds 5 and 6 were found to inhibit tubulin polymerization to the same extent as the anticancer drug vinblastine sulfate, but compounds 11 and 13 inhibited significantly better than vinblastine. Further western blot analysis suggested that compound 6 at 2 μM reduced both levels and phosphorylation state of Akt. Compounds 11 and 13 at 1 μM caused reduced Akt protein levels and strongly suppressed the phosphorylation of Akt. Therefore, 11 and 13 were demonstrated as efficient dual inhibitors of both tubulin polymerization and the Akt pathway and good candidates for further study. More importantly, the strategy of microtubule and Akt dual inhibitors might be a promising direction for developing novel drugs for cancer.

The opioid growth factor-opioid growth factor receptor axis regulates cell proliferation of human hepatocellular cancer

Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide, with a mortality rate approximating its incidence. Understanding the biology of these tumors, as well as treatment modalities, has been challenging. The opioid growth factor (OGF; [Met(5)]-enkephalin) and the OGF receptor (OGFr) form an endogenous growth-regulating pathway in homeostasis and neoplasia. In this investigation, we examined the relationship of the OGF-OGFr axis in HCC and define its presence, function, and mechanism. Using SK-HEP-1, Hep G2, and Hep 3B human HCC cell lines, we found that OGF and OGFr were present and functional. Exogenous OGF was observed to have a dose-dependent, reversible, and receptor-mediated inhibitory action on cell proliferation. Endogenous OGF was found to be constitutively produced and tonically active on cell replicative activities, with neutralization of this peptide accelerating cell proliferation. Silencing of OGFr using siRNA stimulated cell replication, even when exogenous OGF was added to the cultures, documenting its importance in mediating OGF activity. The mechanism of OGF-OGFr action on cell number was related to inhibition of DNA synthesis and not to apoptotic or necrotic pathways. Both OGF and OGFr were detected in surgical specimens of HCC, and no quantitative differences were recorded in peptide or receptor between pathological and normal specimens. These data are the first to report that the OGF-OGFr system is a native biological regulator of cell proliferation in HCC. The findings may provide important insight in designing treatment strategies for this deadly disease.

Regression of established hepatocellular carcinoma is induced by chemoimmunotherapy in an orthotopic murine model.

The high rate of mortality and frequent incidence of recurrence associated with hepatocellular carcinoma (HCC) reveal the need for new therapeutic approaches. In this study we evaluated the efficacy of a novel chemoimmunotherapeutic strategy to control HCC and investigated the underlying mechanism that increased the antitumor immune response. We developed a novel orthotopic mouse model of HCC through seeding of tumorigenic hepatocytes from SV40 T antigen (Tag) transgenic MTD2 mice into the livers of syngeneic C57BL/6 mice. These MTD2‐derived hepatocytes form Tag‐expressing HCC tumors specifically within the liver. This approach provides a platform to test therapeutic strategies and antigen‐specific immune‐directed therapy in an immunocompetent murine model. Using this model we tested the efficacy of a combination of oral sunitinib, a small molecule multitargeted receptor tyrosine kinase (RTK) inhibitor, and adoptive transfer of tumor antigen‐specific CD8+ T cells to eliminate HCC. Sunitinib treatment alone promoted a transient reduction in tumor size. Sunitinib treatment combined with adoptive transfer of tumor antigen‐specific CD8+ T cells led to elimination of established tumors without recurrence. In vitro studies revealed that HCC growth was inhibited through suppression of STAT3 signaling. In addition, sunitinib treatment of tumor‐bearing mice was associated with suppression of STAT3 and a block in T‐cell tolerance. Conclusion: These findings indicate that sunitinib inhibits HCC tumor growth directly through the STAT3 pathway and prevents tumor antigen‐specific CD8+ T‐cell tolerance, thus defining a synergistic chemoimmunotherapeutic approach for HCC. (HEPATOLOGY 2012;55:141–152)

An SV40 VP1-derived epitope recognized by CD8+ T cells is naturally processed and presented by HLA-A*0201 and cross-reactive with human polyomavirus determinants.

The CD8+ T cell responses directed toward the VP1 antigens of human polyomaviruses JC and BK recently were shown to be cross-reactive. Two HLA-A0201-restricted determinants from each virus have been defined and include JCp100-108 (ILMWEAVTL) and BKp108-116 (LLMWEAVTV) as well as JCp36-44 (SITEVECFL) and BKp44-52 (AITEVECFL). We asked whether VP1 from the related SV40 contains similar HLA-A0201-restricted determinants. In this study, we demonstrate that CD8+ T cells specific for SV40 VP1 p110-118 (ILMWEAVTV), but not p46-54 (SFTEVECFL), can be induced in HLA-A0201-transgenic mice and that these CD8+ T cells cross-react with the corresponding determinants from JC and BK virus. The SV40 p110 determinant was found to be processed and presented in SV40-infected cells. These results indicate that the JCp36/BKp44 determinants are distinctive for the human polyomaviruses while the JCp100/BKp108/SVp110 determinants are shared by all three viruses, providing a target for CD8+ T cell cross-reactivity.

A Selenium Containing Inhibitor for the Treatment of Hepatocellular Cancer

Hepatocellular carcinoma (HCC) is the third most deadly cancer in the world. New treatment strategies are desperately needed due to limited standard therapies. Activation of the Erk, Akt, and STAT3pathways is implicated in the prognosis of HCC. The Se,Se′-1,4-phenylenebis(1,2-ethanediyl) bisisoselenourea (PBISe), is a selenium-containing MAPK and PI3 kinase inhibitor, effectively inhibit tumorigenesis in a variety of experimental models. The aim of our study is to demonstrate the potential role of PBISe in the treatment of HCC. The anti-proliferative and pro-apoptotic ability of PBISe is studied in vitro in four human HCC cell lines and in vivo in a spontaneous murine HCC model. Inhibition of cancer growth was performed by cell viability assay and apoptosis by caspase 3/7, PARP cleavage, annexin-V, and TUNEL assays. Role of PBISe on PI3 kinase, MAPK and STAT3 signaling is determined by Western blotting. In vivo effects of PBISe on tumor sizes were monitored using MRI in a spontaneous murine HCC. Liver tissues from the PBISe-treated mice are analyzed for angiogenesis, proliferation, and signaling pathway markers. Overall, PBISe activated caspase-3/7 and increased DNA fragmentation, which is positively correlated with the increased PARP cleavage. PBISe promoted apoptosis by inhibiting PI3K, MAPK, and STAT3 signaling with significant reduction in the tumor sizes (p < 0.007). PBISe-treated tumors reduced survival marker PCNA, and angiogenesis markers Vegf-A, Vegf-R3 and CD34. These results demonstrate the chemotherapeutic effects of PBISe, by inhibiting tumor growth and facilitating tumor apoptosis for HCC treatment.

Abstract 2131: Synthesis and biological evaluation of novel N-alkyl derivatives of 5, 7-dibromoisatin as potent anticancer agents

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Microtubules display an important role in a variety of cellular process, including mitosis and cell division. Various anti-mitotic agents interfering with the natural dynamics of tubulin, polymerization and depolymerization inhibit cancer cell growth. The isatin is found as an endogenous molecule in humans and other mammals, and its analogues display diverse types of biological activities including anti-cancer. Recently, it has been reported that N-alkylation of 5, 7-dibromoisatin increased its cytotoxicity against a range of human cancer cell lines. In this context, it was of interest to further investigate the cytotoxicity of N-alkylated 5,7-dibromoisatin analogues like isothiocyanates and selenocyantes. Isothiocyanates are one of the most effective naturally occurring cancer chemopreventive agents that inhibit carcinogenicity in animal models. Selenium compounds have been found capable of inhibiting and/or retarding tumorigenesis in a variety of experimental animal models. Several synthetic alkyl and aryl selenocyanates have been evaluated for anticarcinogenicity in animal models. The combined literature survey of isatin derivatives, isothiocyantes and selenocyanates have prompted us to develop a novel series of 5,7-dibromoisatin containing thiocyanate, isothiocyanate and selenocyante functionalities. The cytotoxicity of a series of new N-substituted derivatives of 5,7-dibromoisatin was evaluated against a panel of four different human cancer cell lines e.g. a colon (HT29), breast (MCF-7), lung (A549) and melanoma (UACC903) and compared with the 5,7-dibromoisatin. The results showed that the cytotoxicity of the 5,7-dibromoisatin significantly increased through N-alkylation, as reported previously for the 5,7-dibromoisatin derivatives. Introduction of isothiocyante or thiocyanate groups to alkyl chain yielded the most active compounds in this series against HT29 cells. MCF-7 cell line was susceptible to compounds with substitution of selenocyanate group in the alkyl chain, indicating that selenium is playing a role in the antiproliferative activity. These selenium substituted compounds also showed good inhibitory activity against HT29 and A549 cell lines, but poor inhibitory activity in UACC903 cells. Apoptosis assay showed that at 5μM concentration, compounds containing isothiocyanates and thiocyanates induced 99% apoptosis making them the most potent compounds in HT29 cells. The selected compounds were screened for their inhibition of tubulin polymerization in a cell-free in vitro assay. The test compounds significantly inhibited the rate of microtubule polymerization at 10 μM, as compared to vinblastine, a well known microtubule destabilizer. Examination of efficacy of isothiocyanates, thiocyanates, and selenocyanates suggests that these functional groups contribute in enhancing the anticancer activity of novel isatin analogues. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2131. doi:10.1158/1538-7445.AM2011-2131

Abstract 2133: Phenylenebis(1,2-ethanediyl)bisisoselenourea (PBISe), a novel selenium drug for the treatment of hepatocellular cancer (HCC)

Hepatocellular cancer (HCC) is an aggressive tumor that frequently arises in a setting of chronic liver disease and cirrhosis. Appearing 20 to 30 years from initial insult, it is more likely to be discovered in its late stage. The median survival following diagnosis is approximately 6 to 20 months. Although the definitive treatment is tumor resection, most patients are not eligible because of tumor extent or underlying liver dysfunction. Alternative chemopreventive approach in reducing HCC mortality is to be explored. Earlier we have shown the efficacy of PBISe against colon adenocarcinoma and metastatic melanoma with a negligible systemic toxicity in animal studies. In the present study, we intend to demonstrate the chemotherapeutic effects of PBISe on the human HCC. We analyzed the role of PBISe and the underlying mechanism in HCC. PBISe in our study suppressed cell growth, and induce apoptosis. Evaluation of PBISe was performed in vitro on human SK-HEP-1, HepG2, C3A and HUH-7 cells by cellular viability, caspase 3/7 activity, annexin-V expression, DNA fragmentation by Tunnel, cell cycle analysis and AKT, ERK, STAT, SAPK/JNK phosphorylation. PBISe promotes apoptosis by upregulating caspase 3/7, annexin-V, and cPARP. Increased DNA fragmentation was observed with PBISe in all the HCC cell lines tested. PBISe also mediated the cellular proliferation by AKT signaling and downstream pPRAS40 by inducing the cell cycle arrest at G2-M phase. Decreased pERK, pSTAT and pAKT shows that PBISe has a potential to target several signaling pathways. Overall our results suggest that PBISe is a potent chemotherapeutic agent with novel properties for the treatment of HCC. The chemotherapeutic effect of PBISe in the murine HCC model will be discussed. Supported by Penn State Deans Feasibility Grant to H.R.T, NIH RO3 CA 137763-01 to D. D., NIH KO8 417-67HY to K.S.O.C. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2133. doi:10.1158/1538-7445.AM2011-2133