Identification of peptide mimotopes of gp96 using single-chain antibody library.

Heat shock proteins such as gp96 are immunogenic and are widely used as vaccines in immunotherapy of cancers. The present study focuses on the use of peptide mimotopes as immunotherapeutic vaccines for prostate cancer. To this end, we developed a 15-mer gp96 peptide mimotope specifically reactive to MAT-LyLu gp96-peptide complex using combinatorial single-chain antibody and peptide phage display library. The immunogenicity of the synthesized gp96 mimotope was analyzed initially in normal BALB/c mice in combination with various adjuvants such as complete Freund's adjuvant (CFA), aluminum salts (ALUM), granulocyte-macrophage colony-stimulating factor (GM-CSF), and liposome, of which CFA served as a positive control. The antibody response was determined and found that the gp96 mimotope with ALUM showed a significant increase in antibody titer, followed by GM-CSF and liposomes. Further, the T cell (CD4(+) and CD8(+)) populations from splenocytes, as well as IgG isotypes, interleukin-4, and interleukin-5 of gp96 mimotope with ALUM-immunized animals, were analyzed. The results suggest that the gp96 mimotope may elicit a potent and effective antitumor antibody response. Further, the study identifies ALUM and GM-CSF as adjuvant options to drive an appropriate protective immune response as these adjuvants have prior use in humans.

17Beta-estradiol mobilizes bone marrow-derived endothelial progenitor cells to tumors.

Neovascularization is critical for tumor growth and development. The cellular mediators for this process are yet to be defined. We discovered that bone marrow-derived endothelial progenitor cells (BM-EPC), having the phenotype (CD133+, CD34+, VEGFR-2+), initiate neovascularization in response to TG1-1 mammary cells implanted in the inguinal mammary gland of Tie-2 GFP transgenic mice. The fluorescence tag allowed for tracing the migration of green fluorescent protein-tagged endothelial progenitor cells to tumor tissues. We discovered that 17-beta estradiol supplementation of ovariectomized mice significantly enhanced BM-EPC-induced neovascularization and secretion of angiogenic factors within the tumor microenvironment. Cell-based system analyses showed that estrogen-stimulated BM-EPCs secreted paracrine factors which enhanced TG1-1 cell proliferation and migration. Furthermore, TG1-1 cell medium supplemented with estrogen-induced BM-EPC mediated tubulogenesis, which was an experimental in vivo representation of the neovasculature. Our data provide evidence of BM-EPC mammary tumor cell interactions and identify a novel cellular mediator of tumor progression that can be exploited clinically.

Pre-clinical evaluation of 1-nitroacridine derived chemotherapeutic agent that has preferential cytotoxic activity towards prostate cancer.

Chemotherapy in prostate cancer (CaP) even as an adjunct has not been a success. In this communication, we report the pre-clinical efficacy of a nitroacridine derivative, C-1748 (9[2'-hydroxyethylamino]-4-methyl-1-nitroacridine) in CaP cell culture and human xenograft animal models. C-1748, a DNA intercalating agent has been derived from its precursor C-857 that was a potent anti-cancer drug, but failed clinical development due to "high" systemic toxicities. Chemical modifications such as the introduction of a "methyl" group imparted novel properties, the most interesting of which is the difference in the IC(50) values between LnCaP (22.5 nM), a CaP cell line and HL-60, a leukemia cell line (>100 nM). Using gammaH2AX as an intervention marker of DNA double strand breaks, we concluded that C-1748 is more efficacious in CaP cells than in HL-60 cells. In hormone dependent cells, the androgen receptor (AR) was identified as an additional target of C-1748. In xenograft studies, administration of C-1748 intra-peritoneally inhibited tumor growth by 80-90% with minimal toxicity. These studies identify C-1748 as a novel acridine drug that has a high therapeutic index and low cytotoxicity on myelocytic cells with potential for clinical development.

Antiproliferative activity of sulforaphane in Akt-overexpressing ovarian cancer cells.

Epidemiologic studies show a correlation between increased consumption of fruits and vegetables with reduced risk of ovarian cancer. One major bioactive compound found in cruciferous vegetables, particularly broccoli, is sulforaphane, derived from the breakdown of glucoraphanin. We observed potent antiproliferative effects of sulforaphane on human ovarian cancer cell line SKOV3 (IC(50) 40 micromol/L) and mouse ovarian cancer cell lines C3 and T3 (IC(50) 25 micromol/L each) by cell viability assays. The loss of viability is reflected by a down-regulation of cell cycle transition regulators cyclin D1, cyclin-dependent kinase 4 (cdk4), and cdk6. The upstream mediators of sulforaphane effects on the cell cycle in ovarian cancer are still unknown. However, because the Akt signal transduction pathway is overactivated in ovarian cancer, we investigated the effects of sulforaphane on this prosurvival pathway. Both total Akt protein and active phosphorylated levels of Akt (Ser(473)) and phosphoinositide 3-kinase were significantly decreased in sulforaphane-treated SKOV3, C3, and T3 cells with a concomitant inhibition of Akt kinase activity by sulforaphane in SKOV3 and C3 cells. This inhibitory effect of sulforaphane leads to a potent induction of apoptosis in all three cell lines, along with the cleavage of poly(ADP)ribose polymerase. Our study is the first to report the antiproliferative effects of sulforaphane in ovarian cancer and identifying the Akt pathway as a target of sulforaphane, with implications for the inhibition of carcinogenesis by diet-based chemoprevention.

Preclinical toxicological examination of a putative prostate cancer-specific 4-methyl-1-nitroacridine derivative in rodents.

Nitroacridines are potent DNA-binding and cytotoxic agents in cancer cells, but could not be developed clinically due to high systemic toxicities. We are developing a 1-nitroacridine derivative, 9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine (C-1748), as an effective chemotherapeutic agent for prostate cancer. C-1748 demonstrates high antitumor efficacy against human prostate cancer xenografts with markedly low mutagenicity and toxicity in dogs compared with its parent 9-(2'-hydroxyethylamino)-1-nitroacridine (C-857). A surprising feature of C-1748 is the 40-fold difference in 50% inhibitory concentration between DU145 prostate cancer and HL-60 leukemia cells. In this study, we report the preclinical toxicity study of a single acute dose of C-1748 in Copenhagen rats and BALB/c mice, intraperitoneally and intravenously for 24 h and 7 days. The effect of C-1748 on hematology, cardiac and liver enzymes, and renal electrolytes was assessed by blood and serum analysis. The LD50 (lethal dose, 50%) for C-1748 was 9 and 13.42 mg/kg compared with 2.2 and 3 mg/kg for C-857 intraperitoneally and intravenously, respectively, in mice. In Copenhagen rats, LD50 was 15 and 14.4 mg/kg intraperitoneally and intravenously, respectively, compared to 4 and 1.3 mg/kg for C-857. No changes in blood cell counts were observed, which were in the normal range for rodents. No changes were observed in clinical chemistries of enzymes such as aspartate aminotransferase, alkaline phosphatase and creatine phosphokinase, which were within the normal range of values. No genome alterations were seen in prostate cancer cell lines by comparative genomic hybridization together with a lack of systemic toxicity, making it a unique cancer cell-type-specific drug that needs further clinical evaluation for toxicity and synergy in combination chemotherapy regimens.

Synthetic dimer of indole-3-carbinol: second generation diet derived anti-cancer agent in hormone sensitive prostate cancer.

BACKGROUND: Cruciferous vegetables have been found to have anti-prostate cancer effects. The active compounds mediating these effects include indoles such as indole-3-carbinol (I3C) and isothiocyanates. I3C is unstable having tissue tropic effects and clinical utility has been partly addressed by the synthesis of a more stable dimer diindolylmethane (DIM). METHODS: Anti-proliferative activity was measured by XTT assay and cytosolic proteins quantitated by Western blot analysis. RESULTS: DIM (IC(50) 50 microM) is a better anti-proliferative agent than I3C (IC(50) 150 microM) in androgen dependent LNCaP cells, inhibits DNA synthesis, and growth of R1881 stimulated LNCaP cells. Androgen receptor (AR), cyclin D1, and cdk4, induced by R1881, are downregulated by DIM. DIM downregulates phosphorylated Akt and phosphatidyl inositol 3-kinase and downstream inhibition of cyclin D1 and cdk4. CONCLUSION: These studies provide evidence that DIM is a second-generation chemopreventive agent with a viable cellular target and has clinical potential as an anti-prostate cancer chemopreventive.

3,3'-Diindolylmethane downregulates pro-survival pathway in hormone independent prostate cancer.

Epidemiological evidences suggest that the progression and promotion of prostate cancer (CaP) can be modulated by diet. Since all men die with prostate cancer rather than of the disease, it is of particular interest to prevent or delay the progression of the disease by chemopreventive strategies. We have been studying the anticancer properties of compounds present in cruciferous vegetables such as indole-3-carbinol (I3C). Diindolylmethane (DIM) is a dimer of I3C that is formed under acidic conditions and unlike I3C is more stable with higher anti-cancer effects. In the present report, we demonstrate that DIM is a potent anti-proliferative agent compared to I3C in the hormone independent DU 145 CaP cells. The anti-prostate cancer effect is mediated by the inhibition of the Akt signal transduction pathway as DIM, in sharp contrast to I3C, induces the downregulation of Akt, p-Akt, and PI3 kinase. DIM also induced a G1 arrest in DU 145 cells by flow cytometry and downstream concurrent inhibition of cell cycle parameters such as cyclin D1, cdk4, and cdk6. Our data suggest a need for further development of DIM, as a chemopreventive agent for CaP, which justifies epidemiological evidences and molecular targets that are determinants for CaP dissemination/progression. The ingestion of DIM may benefit CaP patients and reduce disease recurrence by eliminating micro-metastases that may be present in patients who undergo radical prostatectomy.

3,3'-Diindolylmethane, a cruciferous vegetable derived synthetic anti-proliferative compound in thyroid disease.

Considerable epidemiological evidence exists to link thyroid disease with differing patterns of dietary consumption, in particular, cruciferous vegetables. We have been studying the anti-thyroid cancer (TCa) activity of indole-3-carbinol (I3C) found in cruciferous vegetables and its acid catalyzed dimer, 3,3'-diindolylmethane (DIM). There are no studies as yet to elucidate the effect of these compounds on the altered proliferative patterns in goiter or thyroid neoplasia. In this study, we tested the anti-proliferative effects of I3C and DIM on four different thyroid cancer cell lines representative of papillary (B-CPAP and 8505-C) and follicular carcinoma of the thyroid (CGTH-W-1 and ML-1), and primary human goiter cells. Cell survival and IC50 values for I3C and DIM were calculated by the XTT assay and cell cycle distribution analysis was done by flow cytometry. DIM was found to be a better anti-proliferative agent than I3C in both papillary and follicular TCa resulting in a greater cytotoxic effect at a concentration over three fold lower than predicted by the molar ratio of DIM and I3C. The anti-proliferative activity of DIM in follicular TCa was mediated by a G1 arrest followed by induction of apoptosis. DIM also inhibited the growth of primary goiter cells by 70% compared to untreated controls. Contrary to traditional belief that cruciferous vegetables are "goitrogenic", DIM has anti-proliferative effects in glandular thyroid proliferative disease. Our preclinical studies provide a strong rationale for the clinical exploration of DIM as an adjuvant to surgery in thyroid proliferative disease.

Differences in glycosylation patterns of heat shock protein, gp96: implications for prostate cancer prevention.

Heat shock protein gp96 induces a tumor-specific protective immunity in a variety of experimental tumor models. Because the primary sequences of the glycoprotein, gp96 are identical between tumor and normal tissues, the peptides associated with gp96 and/or the posttranslational modifications of gp96, determine its immunogenicity. Gp96-associated peptides constitute the antigenic repertoire of the source tissue; thus, purified gp96-peptide complexes have clinical significance as autologous cancer vaccines. However, the role of altered glycosylation and its contribution in the biological as well as immunologic activity of gp96 still remains uncharacterized. We examined the cancer-specific glycosylation patterns of gp96. To this end, monosaccharide compositions of gp96 were compared between normal rat prostate and two cancerous rat prostate tissues, nonmetastatic/androgen-dependent Dunning G and metastatic/androgen-independent MAT-LyLu, as well as two human nonmetastatic prostate cancer cell lines, androgen-dependent LnCaP and androgen-independent DU145. Marked differences were observed between the gp96 monosaccharide compositions of the normal and cancerous tissues. Furthermore, gp96 molecules from more aggressive cellular transformations were found to carry decreasing quantities of several monosaccharides as well as sum total content of neutral and amino sugars. We believe that the unique glycosylation patterns contribute to cellular phenotype and that the posttranslational modifications of gp96 may affect its functional attributes.

Comparative analysis of mutagenic potency of 1-nitro-acridine derivatives.

The anticancer effect of 1-nitro-9-hydroxyethylamino acridine (C-857), a compound belonging to the 1-nitroacridine class, has been well documented. Despite its therapeutic efficacy, the clinical development of C-857 has been impeded partly due to its high systemic toxicity. In an effort to enhance antitumor efficacy and lower toxicity, derivatives of C-857 have been synthesized with substitutions made at position C-4 and/or an esterified hydroxyl group in side chain at the C-9 position. The introduction of a methyl group at C-4 resulted in C-1748, which has a significantly higher therapeutic efficacy and is being clinically developed as an anticancer agent for solid tumors. The present study was undertaken to correlate the mutagenicity of C-857, C-1748, C-1790, C-1872 and C-1873 with their cytotoxicity and their anti-tumor efficacy. The mutagenicity of these drugs was determined using three Ames Salmonella typhimurium strains TA1537, TA98 and TA102. The bacteria were treated with different molar concentrations, ranging from 10(-3) to 10(-12) M, of the drugs and drug-induced histidine revertants were then counted after a 48 h incubation. C-1748 did not induce any revertants in both TA1537 and TA98 at a dose of 10(-6) M, whereas, C-857 at the same dose induced approximately 842 and approximately 1034 revertants respectively. In TA102, mutagenicity was lower than observed with TA98 and TA1537 with highest revertants observed at 10(-5) M with C-857 (approximately 606) and C-1748 (approximately 108). Higher mutagenicity was observed in the derivatives C-1790, C-1872 and C-1873 compared to C-1748, but lower than C-857. These studies demonstrate that C-1748 has the least mutagenic potential, with a much higher antitumor effect in prostate cancer and is a promising chemotherapeutic agent for clinical development.

Aging research in India.

Research on aging in India has been well documented since ancient times. As way back as 3000-1500 BC, the Indian medical system of Ayurveda was used as a means for the prevention of the effects of aging and generation of disease in organs or the whole organism, respectively. In recent years, the focus has been demographic studies on different aspects of aging and has been in isolation. Molecular aspects of aging have been addressed only by a few groups of scientists which has focused on regulation of gene expression, DNA damage and repair, development of immunochemical reagents to detect oxidative DNA damage and assessing the levels of circulating antibodies to reactive oxygen species modified DNA (ROS-DNA), etc. This review aims to recapitulate various research studies on aging since 3000 BC to date.