A new fork for clinical application: Targeting forkhead transcription factors in cancer therapy

RAS-ERK-MAPK (Mitogen-activated protein kinase) pathway plays an essential role in proliferation, differentiation, and tumor progression. In this study, we showed that ERK downregulated FOXO3a through directly interacting with and phosphorylating FOXO3a at Serine 294, Serine 344, and Serine 425. ERK-phosphorylated FOXO3a was degraded by MDM2-mediated ubiquitin-proteosome pathway. FOXO3a phosphorylation and degradation consequently promoted cell proliferation and tumorigenesis. However, the non-phosphorylated FOXO3a mutant, which was resistant to the interaction and degradation by MDM2, resulted in inhibition of tumor formation. Forkhead O transcription factors (FOXOs) are important in the regulation of cellular functions including cell cycle arrest and cell death. Perturbation of FOXOs function leads to deregulated cell proliferation and cancer. Inactivation of FOXO proteins by activation of cell survival pathways, such as PI3K/AKT/IKK, is associated with tumorigenesis. Our study will further highlight FOXOs as new therapeutic targets in a broad spectrum of cancers. ^ Chemotherapeutic drug resistance is the most concerned problem in cancer therapy as resistance ultimately leads to treatment failure of cancer patients. In another study, we showed that blocking ERK activity with AZD6244, an established MEK1/2 inhibitor currently under human cancer clinical trials, enhances FOXO3a expression in various human cancer cell lines in vitro, and also in human colon cancer cell xenografts in vivo. Knocking down FOXO3a and its downstream gene Bim impaired AZD6244-induced growth suppression, whereas restoring activation of FOXO3a sensitized human cancer cell to AZD6244-induced growth arrest and apoptosis. More importantly, AZD6244-resistant cancer cells showed impaired endogenous FOXO3a nuclear translocation, reduced FOXO3a-Bim promoter association and significantly decreased Bim expression in response to AZD6244. AZD6244-resistant cancer cells can be sensitized to API-2 (an AKT inhibitor) and LY294002 (a PI3K inhibitor) in suppressing cell growth and colony formation, these inhibitors were known to enhance FOXO3a activity/nuclear translocation through inhibiting PI3K-AKT pathway. This study reveals novel molecular mechanism contributing to AZD6244-resistance through regulation of FOXO3a activity, further provides significant clinical implication of combining AZD6244 with PI3K/AKT inhibitors for sensitizing AZD6244-resistant cancer cells by activating FOXO3a. FOXO3a activation can be an essential pharmacological target and indicator to mediate and predict AZD6244 efficacy in clinical use. ^

LOW REACTIVE OXYGEN SPECIES AND HIGH GLYCOLYSIS IN GLIOBLASTOMA STEM CELLS:MECHANISMS AND THERAPEUTIC IMPLICATIONS

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor with poor prognosis due in part to drug resistance and high incidence of tumor recurrence. The drug resistant and cancer recurrence phenotype may be ascribed to the presence of glioblastoma stem cells (GSCs), which seem to reside in special stem-cell niches in vivo and require special culture conditions including certain growth factors and serum-free medium to maintain their stemness in vitro. Exposure of GSCs to fetal bovine serum (FBS) can cause their differentiation, the underlying mechanism of which remains unknown. Reactive oxygen species (ROS) play an important role in normal stem cell differentiation, but their role in affecting cancer stem cell fate remains unclear. Whether the metabolic characteristics of GSCs are different from other glioblastoma cells and can be targeted are also unknown. In this study, we used several stem-like glioblastoma cell lines derived from clinical tissues by typical neurosphere culture system or orthotopic xenografts, and showed that addition of fetal bovine serum to the medium induced an increase of ROS, leading to aberrant differentiation and decreases of stem cell markers such as CD133. We found that exposure of GSCs to serum induced their differentiation through activation of mitochondrial respiration, leading to an increase in superoxide (O2-) generation and a profound ROS stress response manifested by upregulation of oxidative stress response pathway. This increase in mitochondrial ROS led to a down-regulation of molecules including SOX2, and Olig2, and Notch1 that are important for stem cell function and an upregulation of mitochondrial superoxide dismutase SOD2 that converts O2- to H2O2. Neutralization of ROS by antioxidant N-acetyl-cysteine in the serum-treated GSCs suppressed the increase of superoxide and partially rescued the expression of SOX2, Olig2, and Notch1, and prevented the serum-induced differentiation phenotype. Additionally, GSCs showed high dependence on glycolysis for energy production. The combination of a glycolytic inhibitor 3-BrOP and a chemotherapeutic agent BCNU depleted cellular ATP and inhibited the repair of BCNU-induced DNA damage, achieving strikingly synergistic killing effects in drug resistant GSCs. This study uncovers the metabolic properties of glioblastoma stem cells and suggests that mitochondrial function and cellular redox status may profoundly affect the fates of glioblastoma stem cells via a ROS-mediated mechanism, and that the active glycolytic metabolism in cancer stem cells may provide a biochemical basis for developing novel therapeutic strategies to effectively eliminate GSCs.

NOVEL THERAPEUTIC TARGETS IDENTIFIED BY HIGH-THROUGHPUT TECHNOLOGIES FOR TRIPLE-NEGATIVE BREAST CANCER

Triple-negative breast cancers (TNBC) are characterized by the lack of or reduced expression of the estrogen and progesterone receptors, and normal expression of the human epidermal growth factor receptor 2. The lack of a well-characterized target for treatment leaves only systemic chemotherapy as the mainstay of treatment. Approximately 60-70% of patients are chemosensitive, while the remaining majority does not respond. Targeted therapies that take advantage of the unique molecular perturbations found in triple-negative breast cancer are needed. The genes that are frequently amplified or overexpressed represent potential therapeutic targets for triple-negative breast cancer. The purpose of this study was to identify and validate novel therapeutic targets for triple-negative breast cancers. 681 genes showed consistent and highly significant overexpression in TNBC compared to receptor-positive cancers in 2 data sets. For two genes, 3 of the 4 siRNAs showed preferential growth inhibition in TNBC cells. These two genes were the low density lipoprotein receptor-related protein 8 (LRP8) and very low-density lipoprotein receptor (VLDLR). Exposure to their cognate ligands, reelin and apolipoprotein E isoform 4 (ApoE4), stimulated the growth of TNBC cells in vitro. Suppression of the expression of either LRP8 or VLDLR or exposure to RAP (an inhibitor of ligand binding to LRP8 and VLDLR) abolished this ligand-induced proliferation. High-throughput protein and metabolic arrays revealed that ApoE4 stimulation rescued TNBC cells from serum-starvation induced up-regulation of genes involved in lipid biosynthesis, increased protein expression of oncogenes involved in the MAPK/ERK and DNA repair pathways, and reduced the serum-starvation induction of biochemicals involved in oxidative stress response and glycolytic metabolism. shLRP8 MDA-MB-231 xenografts had reduced tumor volume, in comparison to parental and shCON xenografts. These results indicate that LRP8-APOE signaling confers survival advantages to TNBC tumors under reduced nutrient conditions and during cellular environmental stress. We revealed that the LRP8-APOE receptor-ligand system is overexpressed in human TNBC. We also demonstrated that this receptor system mediates a strong growth promoting and survival function in TNBC cells in vitro and helps to sustain the growth of MDA-MD-231 xenografts. We propose that inhibitors of LRP8-APOE signaling may be clinically useful therapeutic agents for triple-negative breast cancer.

T-CELL TREATMENTS FOR SOLID AND HEMATOLOGICAL TUMORS

Cell-based therapies have demonstrated potency and efficacy as cancer treatment modalities. T cells can be dichotomized by their T cell receptor (TCR) complexes where alpha/beta T cells (95% of T cells) and gamma/delta T cells (+T cells proliferated to clinically significant numbers and ROR1+ tumor cells were effectively targeted and killed by both ROR1-specific CAR+ T cell populations, although ROR1RCD137 were superior to ROR1RCD28 in clearance of leukemia xenografts in vivo. The second specific aim focused on generating bi-specific CD19-specific CAR+ gamma/delta T cells with polyclonal TCRgamma/delta repertoire on CD19+ artificial antigen presenting cells (aAPC). Enhanced cytolysis of CD19+ leukemia was observed by CAR+ gamma/delta T cells compared to CARneg gamma/delta T cells, and leukemia xenografts were significantly reduced compared to control mice in vivo. The third specific aim looked at the broad anti-tumor effects of polyclonal gamma/delta T cells expanded on aAPC without CAR+ T cells, where Vdelta1, Vdelta2, and Vdelta3 populations had naïve, effector memory, and central memory phenotypes and effector function strength in the following order: Vdelta2>Vdelta3>Vdelta1. Polyclonal gamma/delta T cells eliminated ovarian cancer xenografts in vivo and increased survival compared to control mice. Thus, translating these methodologies to clinical trials will provide cancer patients novel, safe, and effective options for their treatment.

Retinoid -induced regression of human head and neck squamous cell carcinomas is associated with the induction of a pro -inflammatory response

Retinoids are Vitamin A derivatives that are effective chemopreventative and chemotherapeutic agents for head and neck squamous cell carcinomas (HNSCC). Despite the wide application of retinoids in cancer treatment, the mechanism by which retinoids inhibit head and neck squamous cell carcinomas is not completely understood. While in vitro models show that drugs affect cell proliferation and differentiation, in vivo models, such as tumor xenografts in nude mice drugs affect more complex parameters such as extracellular matrix formation, angiogenesis and inflammation. Therefore, we studied the effects of retinoids on the growth of the 22B HNSCC tumors using a xenograft model. In this system, retinoids had no effect on tumor cell differentiation but caused invasion of the tumor by inflammatory cells. Retinoid induced inflammation lead to tumor cell death and tumor regression. Therefore, we hypothesized that retinoids stimulated the 22B HNSCC xenografts to produce a pro-inflammatory signal such as chemokines that in turn activated host inflammatory responses. ^ We used real time quantitative RT-PCR to measure cytokine and chemokine expression in retinoid treated tumors. Treatment of tumors with an RAR-specific retinoid, LGD1550, had no effect on the expression of TNFα, IL-1α, GROα, IP-10, Rantes, MCP-1 and MIP-1α but induced IL-8 mRNA 5-fold. We further characterized the retinoid effect on IL-8 expression on the 22B HNSCC and 1483 HNSCC cells in vitro. Retinoids increased IL-8 expression and enhanced TNFα-dependent IL-8 induction. In addition, retinoids increased the basal and TNFα-dependent expression of MCP-1 but decreased the basal and TNFα dependent expression of IP-10. The effect of retinoids on IL-8 and MCP-1 expression was very rapid with increased levels of mRNA detected within 1–2 hours. This effect did not require new protein synthesis and did not result from mRNA stabilization. Both RAR and RXR ligands increased IL-8 expression whereas only RAR ligands activated MCP-1 expression. ^ We identified a functional retinoid response element in the IL-8 promoter that was located adjacent to the C/EBP-NFkB response element. TNFα treatment of the 22B cells caused rapid, transient and selective acetylation of regions of the IL-8 promoter associated with the NFkB response element. Co-treatment of the cells with retinoids plus TNF increased the acetylation of chromatin in this region without altering the kinetics of acetylation. These results demonstrate that ligand activated retinoid receptors can cooperate with NFkB in histone acetylation and chromatin remodeling. We believe that in certain HNSCC tumors this cooperation and the resulting enhancement of IL-8 expression can induce an inflammatory response that leads to tumor regression. We believe that the induction of inflammation in susceptible tumors, possibly coupled with cytotoxic interventions may be an important component in the use of retinoids to treat human squamous cancers. ^

Upregulation of VEGF through p70S6K in ErbB2 -mediated angiogenesis and inhibition by combined herceptin plus taxol therapy

ErbB2 overexpression in breast tumors increases metastasis, angiogenesis, and reduces survival. To study ErbB2 signaling mechanisms in metastasis and angiogenesis, a spontaneous metastasis assay was performed using human breast cancer cells transfected with constitutively active ErbB2 kinase (V659E), an ErbB2 kinase-dead mutant (K753M), or vector control. Mice injected with V659E had increased metastasis and tumor microvessel density; and the increased angiogenesis in vivo from the V659E transfectants paralleled increased angiogenic potential in vitro, which resulted from increased VEGF by increased protein synthesis. This appeared to be mediated through a PI3K, Akt, mTOR, p70S6K-signaling pathway. Furthermore, V659E xenografts had significantly increased phosphorylated Akt, phosphorylated p70S6K, and VEGF compared with control. To validate the clinical relevance of these findings, human breast tumor samples were examined. Tumors overexpressing ErbB2 correlated with p70S6K phosphorylation and VEGF expression, which significantly correlated with higher levels of Akt and mTOR phosphorylation. Additionally, patients with tumors having increased p70S6K phosphorylation showed a trend for worse disease-free survival and increased metastasis. Together, ErbB2 increases VEGF expression by activating the p70S6K signaling pathway, which may serve as targets for antiangiogenic and antimetastatic therapies. ^ Herceptin is an anti-ErbB2 antibody that demonstrated anti-tumor function, especially in combination with other chemotherapies such as Taxol, in patients with ErbB2-overexpressing tumors. Since the repeated administration of low-dose chemotherapy endorsed an antiangiogenic effect in vitro, and Herceptin was shown to inhibit angiogenesis in tumor xenografts, I investigated whether combined Taxol plus Herceptin treatment inhibits ErbB2-mediated angiogenic responses more effectively. Mice with ErbB2-overexpressing xenografts were treated with control, Herceptin, Taxol, or combination Herceptin plus Taxol. Mice treated with the combination exhibited reduced tumor volumes, tumor microvessel densities, and lung metastasis; and ErbB2-overexpressing cells treated with the combination secreted less VEGF, and stimulated less endothelial cell migration. Furthermore, Akt phosphorylation contributed to VEGF upregulation and was most effectively reduced by combination treatment. ^ In summary, ErbB2 activates signaling to Akt and p70S6K leading to increased VEGF and angiogenesis. Combination Herceptin plus Taxol treatment most effectively inhibited ErbB2-mediated angiogenesis, resulting in pronounced tumoricidal effects, and may be mediated through reduction of phosphorylated Akt, a positive regulator in the p70S6K pathway. ^

p53-independent apoptosis induced by paclitaxel through an indirect mechanism

The efficacy of chemotherapeutic agents may be determined by a number of different factors, including the genotype of the tumor cell. The p53 tumor suppressor gene frequently is mutated in human tumors, and this may contribute to chemotherapeutic resistance. We tested the requirement for wild-type p53 in the response of tumor cells to treatment with paclitaxel (trade name Taxol), an antineoplastic agent that stabilizes cellular microtubules. Although paclitaxel is broadly effective against human tumor xenografts in mice, including some known to carry p53 mutations, we found that p53-containing mouse tumor cells were significantly more sensitive to direct treatment with this drug than were p53-deficient tumor cells. In an attempt to reconcile this apparent discrepancy, we examined the requirement for p53 in the cytotoxic effects of tumor necrosis factor α (TNF-α), a cytokine released from murine macrophages upon paclitaxel treatment. Conditioned medium from paclitaxel-treated macrophages was capable of inducing p53-independent apoptosis when applied to transformed mouse embryonic fibroblasts and was inhibitable by antibodies against TNF-α. Furthermore, in response to direct treatment with TNF-α, both wild-type and p53-deficient tumor cells underwent apoptosis to similar extents and with similar kinetics. Our results suggest that the efficacy of paclitaxel in vivo may be due not only to its microtubule-stabilizing activity, but its ability to activate local release of an apoptosis-inducing cytokine.

A model-based approach for assessing in vivo combination therapy interactions

We present an approach for evaluating the efficacy of combination antitumor agent schedules that accounts for order and timing of drug administration. Our model-based approach compares in vivo tumor volume data over a time course and offers a quantitative definition for additivity of drug effects, relative to which synergism and antagonism are interpreted. We begin by fitting data from individual mice receiving at most one drug to a differential equation tumor growth/drug effect model and combine individual parameter estimates to obtain population statistics. Using two null hypotheses: (i) combination therapy is consistent with additivity or (ii) combination therapy is equivalent to treating with the more effective single agent alone, we compute predicted tumor growth trajectories and their distribution for combination treated animals. We illustrate this approach by comparing entire observed and expected tumor volume trajectories for a data set in which HER-2/neu-overexpressing MCF-7 human breast cancer xenografts are treated with a humanized, anti-HER-2 monoclonal antibody (rhuMAb HER-2), doxorubicin, or one of five proposed combination therapy schedules.

Antitumor activity and pharmacokinetics of a mixed-backbone antisense oligonucleotide targeted to the RIα subunit of protein kinase A after oral administration

Overexpression of the RIα subunit of cAMP-dependent protein kinase (PKA) has been demonstrated in various human cancers. PKA has been suggested as a potential target for cancer therapy. The goal of the present study was to evaluate an anti-PKA antisense oligonucleotide (mixed-backbone oligonucleotide) as a therapeutic approach to human cancer treatment. The identified oligonucleotide inhibited the growth of cell lines of human colon cancer (LS174T, DLD-1), leukemia (HL-60), breast cancer (MCF-7, MDA-MB-468), and lung cancer (A549) in a time-, concentration-, and sequence-dependent manner. In a dose-dependent manner, the oligonucleotide displayed in vivo antitumor activity in severe combined immunodeficient and nude mice bearing xenografts of human cancers of the colon (LS174T), breast (MDA-MB-468), and lung (A549). The routes of drug administration were intraperitoneal and oral. Synergistic effects were found when the antisense oligonucleotide was used in combination with the cancer chemotherapeutic agent cisplatin. The pharmacokinetics of the oligonucleotide after oral administration of 35S-labeled oligonucleotide into tumor-bearing mice indicated an accumulation and retention of the oligonucleotide in tumor tissue. This study further provides a basis for clinical studies of the antisense oligonucleotide targeted to the RIα subunit of PKA (GEM 231) as a cancer therapeutic agent used alone or in combination with conventional chemotherapy.

Growth hormone-releasing hormone: An autocrine growth factor for small cell lung carcinoma

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the growth of various cancers in vivo. This effect is thought to be exerted through suppression of the pituitary growth hormone–hepatic insulin-like growth factor I (IGF-I) axis and direct inhibition of autocrine/paracrine production of IGF-I and -II in tumors. However, other evidence points to a direct effect of GHRH antagonists on tumor growth that may not implicate IGFs, although an involvement of GHRH in the proliferation of cancer cells has not yet been established. In the present study we investigated whether GHRH can function as an autocrine/paracrine growth factor in small cell lung carcinoma (SCLC). H-69 and H-510A SCLC lines cultured in vitro express mRNA for GHRH, which apparently is translated into peptide GHRH and then secreted by the cells, as shown by the detection of GHRH-like immunoreactivity in conditioned media from the cells cultured in vitro. In addition, the levels of GHRH-like immunoreactivity in serum from nude mice bearing H-69 xenografts were higher than in tumor-free mice. GHRH(1–29)NH2 stimulated the proliferation of H-69 and H-510A SCLCs in vitro, and GHRH antagonist JV-1–36 inhibited it. JV-1–36 administered s.c. into nude mice bearing xenografts of H-69 SCLC reduced significantly (P < 0.05) tumor volume and weight, after 31 days of therapy, as compared with controls. Collectively, our results suggest that GHRH can function as an autocrine growth factor in SCLCs. Treatment with antagonistic analogs of GHRH may offer a new approach to the treatment of SCLC and other cancers.

Synergistic inhibition of human cancer cell growth by cytotoxic drugs and mixed backbone antisense oligonucleotide targeting protein kinase A

Protein kinase A type I plays a key role in neoplastic transformation, conveying mitogenic signals of different growth factors and oncogenes. Inhibition of protein kinase A type I by antisense oligonucleotides targeting its RIα regulatory subunit results in cancer cell growth inhibition in vitro and in vivo. A novel mixed backbone oligonucleotide HYB 190 and its mismatched control HYB 239 were tested on soft agar growth of several human cancer cell types. HYB 190 demonstrated a dose-dependent inhibition of colony formation in all cell lines whereas the HYB 239 at the same doses caused a modest or no growth inhibition. A noninhibitory dose of each mixed backbone oligonucleotide was used in OVCAR-3 ovarian and GEO colon cancer cells to study whether any cooperative effect may occur between the antisense and a series of cytotoxic drugs acting by different mechanisms. Treatment with HYB 190 resulted in an additive growth inhibitory effect with several cytotoxic drugs when measured by soft agar colony formation. A synergistic growth inhibition, which correlated with increased apoptosis, was observed when HYB 190 was added to cancer cells treated with taxanes, platinum-based compounds, and topoisomerase II selective drugs. This synergistic effect was also observed in breast cancer cells and was obtained with other related drugs such as docetaxel and carboplatin. Combination of HYB 190 and paclitaxel resulted in an accumulation of cells in late S-G2 phases of cell cycle and marked induction of apoptosis. A cooperative effect of HYB 190 and paclitaxel was also obtained in vivo in nude mice bearing human GEO colon cancer xenografts. These results are the first report of a cooperative growth inhibitory effect obtained in a variety of human cancer cell lines by antisense mixed backbone oligonucleotide targeting protein kinase A type I-mediated mitogenic signals and specific cytotoxic drugs.

Cytokine-mediated enhancement of epidermal growth factor receptor expression provides an immunological approach to the therapy of pancreatic cancer

The increased expression of epidermal growth factor receptor induced by tumor necrosis factor α renders pancreatic cancer cells more susceptible to antibody-dependent cellular cytotoxicity by a mAb specific for this receptor. Laboratory studies with athymic mice bearing xenografts of human pancreatic cancer cells demonstrated a cytokine-induced ability of the mAb to cause significant tumor regression. In a phase I/II clinical trial, 26 patients with unresectable pancreatic cancer were enrolled into three cohorts receiving variable amounts of the antibody together with a constant amount of tumor necrosis factor α. With increasing doses of antibody, the growth of the primary tumor was significantly inhibited. This was reflected by a longer median survival, with one complete remission lasting for 3 years obtained with the highest dose of antibody employed. Thus, a combination of the cytokine, tumor necrosis factor α, with a mAb to the epidermal growth factor receptor offers a potentially useful approach for the treatment of pancreatic cancer.

Production of infectious bovine papillomavirus from cloned viral DNA by using an organotypic raft/xenograft technique

Bovine papillomavirus type 1 (BPV-1) induces fibropapillomas in its natural host and can transform fibroblasts in culture. The viral genome is maintained as an episome within fibroblasts, which has allowed extensive genetic analyses of the viral functions required for DNA replication, gene expression, and transformation. Much less is known about BPV-1 gene expression and replication in bovine epithelial cells because the study of the complete viral life cycle requires an experimental system capable of generating a fully differentiated stratified bovine epithelium. Using a combination of organotypic raft cultures and xenografts on nude mice, we have developed a system in which BPV-1 can replicate and produce infectious viral particles. Organotypic cultures were established with bovine keratinocytes plated on a collagen raft containing BPV-1-transformed fibroblasts. These keratinocytes were infected with virus particles isolated from a bovine wart or were transfected with cloned BPV-1 DNA. Several days after the rafts were lifted to the air interface, they were grafted on nude mice. After 6–8 weeks, large xenografts were produced that exhibited a hyperplastic and hyperkeratotic epithelium overlying a large dermal fibroma. These lesions were strikingly similar to a fibropapilloma caused by BPV-1 in the natural host. Amplified viral DNA and capsid antigens were detected in the suprabasal cells of the epithelium. Moreover, infectious virus particles could be isolated from these lesions and quantitated by a focus formation assay on mouse cells in culture. Interestingly, analysis of grafts produced with infected and uninfected fibroblasts indicated that the fibroma component was not required for productive infection or morphological changes characteristic of papillomavirus-infected epithelium. This system will be a powerful tool for the genetic analysis of the roles of the viral gene products in the complete viral life cycle.

Combined effect of tumor necrosis factor-related apoptosis-inducing ligand and ionizing radiation in breast cancer therapy

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent endogenous activator of the cell death pathway and functions by activating the cell surface death receptors 4 and 5 (DR4 and DR5). TRAIL is nontoxic in vivo and preferentially kills neoplastically transformed cells over normal cells by an undefined mechanism. Radiotherapy is a common treatment for breast cancer as well as many other cancers. Here we demonstrate that ionizing radiation can sensitize breast carcinoma cells to TRAIL-induced apoptosis. This synergistic effect is p53-dependent and may be the result of radiation-induced up-regulation of the TRAIL-receptor DR5. Importantly, TRAIL and ionizing radiation have a synergistic effect in the regression of established breast cancer xenografts. Changes in tumor cellularity and extracellular space were monitored in vivo by diffusion-weighted magnetic resonance imaging (diffusion MRI), a noninvasive technique to produce quantitative images of the apparent mobility of water within a tissue. Increased water mobility was observed in combined TRAIL- and radiation-treated tumors but not in tumors treated with TRAIL or radiation alone. Histological analysis confirmed the loss of cellularity and increased numbers of apoptotic cells in TRAIL- and radiation-treated tumors. Taken together, our results provide support for combining radiation with TRAIL to improve tumor eradication and suggest that efficacy of apoptosis-inducing cancer therapies may be monitored noninvasively, using diffusion MRI.

Gonadotropin-releasing hormone analogue conjugates with strong selective antitumor activity

Conjugation of gonadotropin-releasing hormone (GnRH) analogues GnRH-III, MI-1544, and MI-1892 through lysyl side chains and a tetrapeptide spacer, Gly-Phe-Leu-Gly (X) to a copolymer, poly(N-vinylpyrrolidone-co-maleic acid) (P) caused increased antiproliferative activity toward MCF-7 and MDA-MB-231 breast, PC3 and LNCaP prostate, and Ishikawa endometrial cancer cell lines in culture and against tumor development by xenografts of the breast cancer cells in immunodeficient mice. MCF-7 cells treated with P-X-1544 and P-X-1892 displayed characteristic signs of apoptosis, including vacuoles in the cytoplasm, rounding up, apoptotic bodies, bleb formation, and DNA fragmentation. Conjugates, but not free peptides, inhibited cdc25 phosphatase and caused accumulation of Ishikawa and PC3 cells in the G2/M phase of the cell cycle after 24 h at lower doses and in the G1 and G2 phases after 48 h. Since P-X-peptides appear to be internalized, the increased cytotoxicity of the conjugates is attributed to protection of peptides from proteolysis, enhanced interaction of the peptides with the GnRH receptors, and/or internalization of P-X-peptide receptor complexes so that P can exert toxic effects inside, possibly by inhibiting enzymes involved in the cell cycle. The additional specificity of P-X-peptides compared with free peptides for direct antiproliferative effects on the cancer cells but not for interactions in the pituitary indicates the therapeutic potential of the conjugates.