Supplementary Figure 1 from 17β-Estradiol Mobilizes Bone Marrow–Derived Endothelial Progenitor Cells to Tumors

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.

AbstractNeovascularization 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-β 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. [Cancer Res 2008;68(15):6038–42]

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 (IC50 40 μmol/L) and mouse ovarian cancer cell lines C3 and T3 (IC50 25 μmol/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 (Ser473) 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. [Mol Cancer Ther 2007;6(1):334–45]

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 IC50 values between LnCaP (22.5 nM), a CaP cell line and HL-60, a leukemia cell line (> 100 nM). Using gamma H2AX 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.

4780 Autologous HSP96 is being evaluated as a multivalent peptide cancer vaccine. The ability of HSP96 to act as a peptide chaperone forms the basis for its use as a multivalent biological cancer vaccine. Internalization of HSP96/peptide complex occurs through cell surface receptors, such as CD91 resulting in cross presentation of cancer specific antigenic peptides and elicitation of tumor specific cytotoxic T lymphocytes (CTLs). HSP96 is a glycoprotein containing two known N-linked glycan structures and four more potential N-linked glycosylation sites. However, the role of these glycosyl moieties, if any, on the structure and function of HSP96 is currently unknown. We discovered that purified HSP96, from various human and rat prostate tumors have significant differences in glycosylation patterns which is correlated with cell specific phenotype and removal of glycosyl moieties modulates peptide binding affinity. We demonstrated these effects using glycosylated and unglycosylated purified HSP96 and VSV-8 peptide (RGYVYQGL). These results for the first time implicate glycans as signatorial elements of tissue derived HSP96 that modulate its peptide chaperoning and binding ability. Other functions of HSP96 such as the integral ATPase activity and cell surface receptor mediated endocytosis may also be dependent on glycan structures. These studies are intended to define the structural components that determine the pleomorphic functions of HSP96 based cancer vaccines.

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.

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.

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.

Proc Amer Assoc Cancer Res, Volume 46, 2005 2460 Compounds in cruciferous vegetables such as indole-3-carbinol (I3C) have a significant role in prevention of many different cancers. However, the anti-proliferative activity of these compounds in thyroid cancer has not been documented. We have been studying the anti-thyroid cancer (CaT) activity of indole-3-carbinol (I3C) and its acid catalyzed dimer, diindolylmethane (DIM) using four different cell lines representing papillary cancer (8505-C and B-CPAP) and follicular carcinoma of the thyroid (ML-1 and CGTHW-1). Cell survival and IC50 values were calculated using the XTT assay and the Trypan Blue exclusion test. Apoptosis was measured by ELISA and protein levels were estimated by western blotting. Both I3C and DIM showed significant anti-CaT effects. I3C had IC50 values that ranged between 100-500 μM, whereas, the IC50 values for DIM were significantly lower between 10-100 μM. The anti-proliferative effect observed with I3C and DIM was due to induction of apoptosis; however, apoptosis induced by DIM was greater than that seen with I3C. Microscopic examination of primary culture of cells from a goiter nodule revealed a significant reduction in the number of DIM and I3C treated cells accompanied by morphological changes. The effects of I3C and DIM on signal transduction pathways were investigated as possible targets for the observed anti-cancer effect. Western blot analysis revealed that the Akt signal transduction pathway is a target of both I3C and DIM. PI3-K and p-Akt were markedly down regulated in ML-1, CGTHW-1 and B-CPAP. The results clearly indicate that DIM has a higher anti-proliferative activity compared to I3C. Based on these data we have initiated a human clinical trial to examine the distribution and retention of DIM in thyroid tissues.

5885 Development of a chemotherapeutic regimen against prostate cancer (CaP) is a challenge. We have identified a novel nitroacridine, C-1748 (Capridine-β) that has pronounced antitumor activity in human prostate cancer xenograft models and its activity was comparable in both hormone dependent (LnCaP) and hormone independent (DU-145) models. In an effort to delineate the molecular targets of C-1748, we found a marked induction of androgen receptor (AR) in C-1748 treated hormone independent DU-145 cells. The induction of the androgen receptor was observed starting at 6 hours after treatment with 5 nM C-1748 and continued to increase till 24 hours as measured by western blot analysis. These observations were further confirmed by immunoflourescence studies which showed a dose dependent enhancement of AR in response to C-1748 in DU-145 cells. We also examined the steady state level of ERβ which showed a dose dependent down regulation consistent with the anticancer effect of ERβ in prostate cancer. The functional consequence of C-1748 mediated AR induction in hormone independent CaP was analyzed by examining the synergistic effect of the anti-androgen flutamide and C-1748 on DU-145 and LnCaP cells by XTT assays. We observed marked synergistic activity between C-1748 and flutamide in DU-145 cells suggesting that the andogen receptor induced by C-1748 was functional. In LnCaP, flutamide inhibited cell proliferation but no synergism with C-1748 was observed. We conclude that androgen receptor mediated signal transduction is one of the targets of C-1748 that is presumably restricted to hormone independent cells. This implies that C-1748 can be incorporated synergistically in an antiandrogen based treatment regimen for hormone independent, highly aggressive prostate cancer.

1567 Epidemiological studies have shown that an increased consumption of fruits and vegetables reduces the risk of ovarian cancer. However, the literature does not contain many reports on the use of dietary components as chemopreventive agents for ovarian cancer. Sulforaphane (SFN) is derived from the breakdown of glucoraphanin and is found in large amounts in cruciferous vegetables, particularly broccoli. We observed a strong anti-cancer effect of SFN on human ovarian cancer cell line SKOV3 by cell viability assays and determined the IC50 to be 10 μM. The loss of viability was due to induction of apoptosis where we observed a four fold increase in apoptosis at 10 μM going upto 20 fold at 100 μM. The apoptotic effect that results in the loss of viability is preceded by a significant effect on the regulator of cell cycle transition molecules specifically cdk4. The steady state level of cdk4 decreased in dose dependent manner with complete abrogation at 20 μM. In an effort to identify further upstream targets of SFN we examined the effect of SFN on signal transduction pathways that may be of significance in ovarian cancer. We observed that both total Akt protein and the active phosphorylated levels of Akt were significantly inhibited in SFN treated SKOV3 cells. We conclude that the pro-survival Akt pathway is a target of the chemopreventive SFN that may lead to cell cycle inhibition and induction of apoptosis and propose use of SFN with other chemotherapeutic agents that target phosphorylation mediated signal transduction pathways in ovarian cancer.

Proc Amer Assoc Cancer Res, Volume 45, 20043034 Aromatase inhibitors (anastrozole, letrozole, and exemestane) are considered viable alternatives to tamoxifen for breast cancer therapy because they work by blocking the conversion of androgens to estrogen in post-menopausal women. Indeed, anastrozole has been shown to be superior to tamoxifen for the first-line treatment of postmenopausal women with advanced breast cancer, and letrozole is being investigated in several large adjuvant trials. However, there are concerns that like tamoxifen, patients who initially respond to aromatase inhibitors will eventually develop resistance. To address the issue of acquired resistance to aromatase inhibitors, we have developed a hormone-independent breast cancer cell line, MCF7:5C, which was originally cloned from the wild-type MCF7 cells [Jiang et al ., Mol. Cell. Endocrinol. 1992., 90(1):77-86]. Using DNA quantitation and trypan blue exclusion assays, we examined the effects of E2 and the antiestrogens 4-hydroxytamoxifen (4OHT), raloxifene, and fulvestrant on the growth of MCF7:5C cells. Our results showed that while E2 treatment (1 nM) stimulated the growth of parental MCF7 cells (∼10-fold above control), it caused a dramatic reduction in the growth of MCF7:5C cells with maximum inhibition (∼90%) at day 7. 4OHT, raloxifene, and fulvestrant (at 1 μM) also reduced the growth of MCF7:5C cells (55%, 62%, and 80%, respectively), however, their inhibitory effects were less than that of E2. To investigate whether the E2-induced growth inhibition of MCF7:5C cells was due to apoptosis, analyses were performed by Annexin V-FITC/Propidium iodide and DAPI staining and confirmed by electron microscopy. Flow cytometry analysis of MCF7:5C cells revealed that apoptotic cell population increased gradually from less than 5% in vehicle control to ∼70-80% with 1 nM E2, and that this effect was almost completely blocked by the universal capase inhibitor z-VAD-FMK and fulvestrant. Interestingly, we found that fulvestrant did not induce apoptosis in MCF7:5C cells despite its ability to significantly reduce the growth of these cell, thus suggesting an alternative mechanism for its growth inhibitory effects. Furthermore, western blot analyses revealed that E2 treatment significantly reduced the levels of the anti-apoptotic proteins Bcl-2, Bcl-xl, and Hsp27 but significantly increased the levels of Bax, Bid, and Bak (pro-apoptotic). Overall, these results show that E2 is capable of inducing apoptosis in long-term estrogen-deprived breast cancer cells and that this effect is most likely capase dependent and ER-mediated. This research was supported by the DOD grant DAMD17-001-0386 and the signal transduction grant T32CA70085.