Engineering the first chimeric antibody in targeting intracellular PRL-3 oncoprotein for cancer therapy in mice.

Antibodies are considered as 'magic bullets' because of their high specificity. It is believed that antibodies are too large to routinely enter the cytosol, thus antibody therapeutic approach has been limited to extracellular or secreted proteins expressed by cancer cells. However, many oncogenic proteins are localized within the cell. To explore the possibility of antibody therapies against intracellular targets, we generated a chimeric antibody targeting the intracellular PRL-3 oncoprotein to assess its antitumor activities in mice. Remarkably, we observed that the PRL-3 chimeric antibody could efficiently and specifically reduce the formation of PRL-3 expressing metastatic tumors. We further found that natural killer (NK) cells were important in mediating the therapeutic effect, which was only observed in a nude mouse model (T-cell deficient), but not in a Severe Combined Immunodeficiency' (scid ) mouse model (B- and T-cell deficient), indicating the anticancer effect also depends on host B-cell activity. Our study involving 377 nude and scid mice suggest that antibodies targeting intracellular proteins can be developed to treat cancer.

Targeting intracellular oncoproteins with antibody therapy or vaccination.

Antibody-based therapies have better specificity and thus improved efficacy over standard chemotherapy regimens, which result in extended survival and improved quality of life for cancer patients. Because antibodies are viewed as too large to access intracellular locations, antibody therapy has traditionally targeted extracellular or secreted proteins expressed by cancer cells. However, many oncogenic proteins are found within the cell (such as intracellular phosphatases/kinases and transcription factors) and have therefore not been pursued for antibody therapies. Here, we explored the possibility of antibody therapy or vaccination against intracellular proteins. As proofs of concept, we selected three representative intracellular proteins as immunogens for tumor vaccine studies: PRL-3 (phosphatase of regenerating liver 3), a cancer-associated phosphatase; EGFP (enhanced green fluorescent protein), a general reporter; and mT (polyomavirus middle T), the polyomavirus middle T oncoprotein. A variety of tumors that expressed these intracellular proteins were clearly inhibited by their respective exogenous antibodies or by antigen-induced host antibodies (vaccination). These anticancer activities were reproducibly observed in hundreds of C57BL/6 tumor-bearing mice and MMTV-PymT transgenic breast tumor mice. Our in vivo data suggest that immunotherapies can target not only extracellular but also intracellular oncoproteins.

Dietary feeding of silibinin inhibits prostate tumor growth and progression in transgenic adenocarcinoma of the mouse prostate model.

Herein, for the first time, we evaluated the chemopreventive efficacy of dietary silibinin against prostate cancer (PCa) growth and progression in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice from two different genetic backgrounds [C57BL/6 (TRAMP) x FVB; C57BL/6 (TRAMP) x C57BL/6]. At 4 weeks of age, mice were fed control or 0.1% to 1% silibinin-supplemented diets until 23 to 24 weeks of age. Silibinin-fed groups had a lower tumor grade and higher incidence of prostatic intraepithelial neoplasia (PIN) at the expense of a strong decrease in adenocarcinoma incidence. Prostate tissue showed a 47% (P < 0.001) decrease in proliferating cell nuclear antigen (PCNA)-positive cells and an approximately 7-fold (P < 0.001) increase in apoptotic cells at the highest silibinin dose. As potential mechanisms of silibinin efficacy, an approximately 50% (P < 0.05) decrease in insulin-like growth factor (IGF) receptor type I beta and an approximately 13-fold (P < 0.001) increase in IGF-binding protein 3 (IGFBP-3) protein levels were also observed. These changes were specific to tumors as they were not reflected in circulating IGF-IGFBP-3 system. Additionally, silibinin decreased protein expression of cyclin-dependent kinases (Cdk) by more than 90% (P < 0.001) with a concomitant increase in Cdk inhibitors, Cip1/p21 and Kip1/p27 (P < 0.05, for both). A dose-dependent decrease was also observed in cyclin B1, cyclin E, and cyclin A protein levels by silibinin. Together, these findings suggest that oral silibinin blocks PCa growth and progression at PIN stage in TRAMP mice via modulation of tumor IGF-IGFBP-3 axis and cell cycle regulation, and therefore it has practical and translational potential in suppressing growth and neoplastic conversion of PIN to PCa in humans.

Silibinin efficacy against human hepatocellular carcinoma.

PURPOSE: Hepatocellular carcinoma (HCC) is one of the most common recurrent malignancies, for which, currently, there is no effective therapy. Considering the antihepatotoxic activity of silibinin, a widely used drug and supplement for various liver disorders, together with its strong preventive and anticancer efficacy against various epithelial cancers, we investigated the efficacy of silibin against human HCC cells. EXPERIMENTAL DESIGN: Silibinin effects were examined on growth, cytotoxicity, apoptosis, and cell cycle progression in two different HCC cell lines, HepG2 (hepatitis B virus negative; p53 intact) and Hep3B (hepatitis B virus positive; p53 mutated). At molecular level, cell cycle effects of silibinin were assessed by immunoblotting and in-bead kinase assays. RESULTS: Silibinin strongly inhibited growth of both HepG2 and Hep3B cells with a relatively stronger cytotoxicity in Hep3B cells, which was associated with apoptosis induction. Silibinin also caused G1 arrest in HepG2 and both G1 and G2-M arrests in Hep3B cells. Mechanistic studies revealed that silibinin induces Kip1/p27 but decreases cyclin D1, cyclin D3, cyclin E, cyclin-dependent kinase (CDK)-2, and CDK4 levels in both cell lines. In Hep3B cells, silibinin also reduced the protein levels of G2-M regulators. Furthermore, silibinin strongly inhibited CDK2, CDK4, and CDC2 kinase activity in these HCC cells. CONCLUSION: Together, these results for the first time identify the biological efficacy of silibinin against HCC cells, suggesting the importance of conducting further investigations in preclinical HCC models, especially on in vivo efficacy, to support the clinical usefulness of silibinin against hepatocellular carcinoma in addition to its known clinical efficacy as an antihepatotoxic agent.

Antioxidant activity of Smoke Shield in-vitro and in-vivo.

Smoke Shield is a proprietory formulation containing extract of turmeric (Curcuma longa), obtained by supercritical carbon dioxide gas extraction and post-supercritical hydroethanolic extraction, together with extracts of green tea and other spices whose presence synergistically increases the activity of turmeric. This study evaluates the antioxidant potentials of Smoke Shield in-vitro and in experimental animals, as well as in human models. Smoke Shield was found to scavenge superoxide radicals generated by photoreduction of riboflavin (50% inhibitory concentration = 91 microg mL(-1)) and hydroxyl radicals generated by Fenton reaction (50% inhibitory concentration = 95 microg mL(-1)) and reduced lipid peroxidation. Administration of Smoke Shield to mice was found to elevate antioxidant enzymes such as catalase and superoxide dismutase in blood as well as in liver and kidney. Glutathione-S-transferase activity was found to be significantly elevated in liver and kidney of animals treated with Smoke Shield. Glutathione levels were also significantly elevated in blood. Glutathione reductase was significantly elevated in kidney. Administration of Smoke Shield decreased the lipid peroxidation in serum, liver and kidney, as well as reduced the levels of conjugated dienes and hydroperoxides. Administration of Smoke Shield to smokers was found to increase the superoxide dismutase and glutathione in blood and decrease glutathione peroxidase. Smoke Shield inhibited phase I enzymes as represented by aniline-hydroxylase and aminopyrenedemethylase in-vitro. These results indicate that Smoke Shield has potent antioxidant activity, could inhibit phase I enzymes and increase detoxifying enzymes, which makes it an effective chemoprotective herbal formulation.