In vivo evaluation of safety of nanoporous silicon carriers following single and multiple dose intravenous administrations in mice.

Porous silicon (pSi) is being extensively studied as an emerging material for use in biomedical applications, including drug delivery, based on the biodegradability and versatile chemical and biophysical properties. We have recently introduced multistage nanoporous silicon microparticles (S1MP) designed as a cargo for nanocarrier drug delivery to enable the loaded therapeutics and diagnostics to sequentially overcome the biological barriers in order to reach their target. In this first report on biocompatibility of intravenously administered pSi structures, we examined the tolerability of negatively (-32.5±3.1mV) and positively (8.7±2.5mV) charged S1MP in acute single dose (10(7), 10(8), 5×10(8) S1MP/animal) and subchronic multiple dose (10(8) S1MP/animal/week for 4 weeks) administration schedules. Our data demonstrate that S1MP did not change plasma levels of renal (BUN and creatinine) and hepatic (LDH) biomarkers as well as 23 plasma cytokines. LDH plasma levels of 145.2±23.6, 115.4±29.1 vs. 127.0±10.4; and 155.8±38.4, 135.5±52.3 vs. 178.4±74.6 were detected in mice treated with 10(8) negatively charged S1MP, 10(8) positively charged S1MP vs. saline control in single and multiple dose schedules, respectively. The S1MPs did not alter LDH levels in liver and spleen, nor lead to infiltration of leukocytes into the liver, spleen, kidney, lung, brain, heart, and thyroid. Collectively, these data provide evidence of a safe intravenous administration of S1MPs as a drug delivery carrier.

Dynamic change in phosphorylated platelet-derived growth factor receptor in peripheral blood leukocytes following docetaxel therapy predicts progression-free and overall survival in prostate cancer.

In a placebo-controlled randomised study of the platelet-derived growth factor receptor (PDGFR) inhibitor imatinib mesylate and docetaxel in metastatic prostate cancer with bone metastases (n=116), no significant differences in progression-free and overall survival were observed. To evaluate pharmacodynamic correlates of outcomes, we assessed the association of plasma platelet-derived growth factor (PDGF) isoform kinetics and PDGFR inhibition with progression-free and overall survival by individual treatment arm. We found that in the docetaxel-placebo arm alone, the probability of decrease in PDGFR phosphorylation (Pr-Decr-pPDGFR) above 0.5 (vs 30 months (HR 3.1; P=0.04 in log-rank test). By contrast, in the docetaxel plus imatinib arm, the association of Pr-Decr-pPDGFR >0.5 with a rise in plasma PDGF isoform concentrations and inferior survival was not observed. The data suggest that dynamic changes in PDGFR phosphorylation in peripheral blood leukocytes predict docetaxel efficacy. Rising plasma PDGF concentrations may explain and/or mark docetaxel resistance. Validation and mechanistic studies addressing these unexpected findings should anticipate a confounding influence of concurrent PDGFR inhibitor therapy.

Dual inhibition of the epidermal growth factor and vascular endothelial growth factor phosphorylation for antivascular therapy of human prostate cancer in the prostate of nude mice.

BACKGROUND: Androgen-independent prostate cancer (PCa) may be susceptible to modulation of the tumor microenvironment. We determined whether a dual tyrosine kinase inhibitor (AEE788) of the epidermal growth factor receptor (EGF-R) and vascular endothelial growth factor receptor (VEGF-R) combined with chemotherapy can produce therapy of human PCa in nude mice. METHODS: PC-3MM2 human PCa cells were injected into the prostate of nude mice. Three days later, the mice were randomized into four groups: saline control, paclitaxel, AEE788, and AEE788 and paclitaxel. The mice were treated for 5 weeks and necropsied. Tumor incidence, weight, and incidence of lymph node metastasis were recorded. Tumor tissue was analyzed immunohistochemically. RESULTS: Treatment of mice with AEE788 or AEE788 plus paclitaxel significantly decreased tumor incidence, total tumor weight, and incidence of lymph node metastasis. AEE788 treatment alone or in combination with paclitaxel inhibited the phosphorylation of EGF-R and VEGF-R on tumor cells and tumor-associated endothelial cells. Therapeutic efficacy correlated with an increase in apoptosis of tumor cells and tumor-associated endothelial cells. CONCLUSION: Blockade of EGF-R and VEGF-R signaling pathways coupled with chemotherapy suppressed the progressive growth and metastasis of human PCa cells growing orthotopically in nude mice.

Epidermal hyperplasia overlying human melanoma correlates with tumour depth and angiogenesis.

The aim of this study was to determine whether epidermal hyperplasia overlying cutaneous human melanoma is associated with increased tumour angiogenesis, tumour growth and the potential for metastasis. Forty-two surgical specimens of cutaneous human melanoma of different depths, each containing epidermis present in the tumour-free margin, were analysed by immunohistochemistry for the expression of the pro-angiogenic molecules basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) and the anti-angiogenic molecule interferon-beta (IFN-beta). The epidermis overlying intermediate and thick (1.0-10.0 mm), but not thin (0.5-1.0 mm), melanoma specimens was hyperplastic. Although the expression level of bFGF, VEGF and IL-8 in the epidermis directly overlying the tumour was similar to that in the distant epidermis, the expression of IFN-beta was significantly decreased in keratinocytes overlying intermediate and thick, but not thin, melanomas. The microvessel density was also increased in intermediate and thick specimens. Human melanoma cells were injected subcutaneously into nude mice. The resulting tumours were used to determine the association between overlying epidermal hyperplasia and neoplastic angiogenesis. Similar to human autochthonous melanomas, epidermal hyperplasia was found only over lesions produced by metastatic cells. Although there was no change in the expression of the pro-angiogenic molecules, the expression of IFN-beta was significantly decreased in the hyperplastic epidermis. Conditioned medium collected from cultures of the metastatic cell line induced in vitro proliferation of mouse keratinocytes, whereas conditioned medium collected from cultures of the non-metastatic cell line did not. Collectively, the data demonstrate that metastatic melanoma cells induce keratinocyte proliferation, leading to decreased expression of the negative regulator of angiogenesis, IFN-beta, and hence to increased angiogenesis.

Interferon-alpha prevents selection of doxorubicin-resistant undifferentiated-androgen-insensitive metastatic human prostate cancer cells.

BACKGROUND: We determined whether treatment of metastatic prostate cancer cells with doxorubicin (DOX) and interferon-alpha (IFN-alpha) prevented the emergence of highly undifferentiated tumor cells. METHODS: The state of cell differentiation was determined by analysis of prostate-specific antigen (PSA), E-cadherin, keratin, and vimentin. RESULTS: Human prostate cancer LNCaP-LN3 cells growing in culture as multicell spheroids expressed higher levels of E-cadherin and E-cadherin-associated beta-catenin than LNCaP-LN3 cells growing as monolayers. Treatment of cells with DOX downregulated PSA, E-cadherin, and keratin, and upregulated expression of vimentin and vascular endothelial growth factor (VEGF) mRNA. While treatment of cells with IFN-alpha did not alter gene expression, the addition of IFN-alpha to cultures treated with DOX produced synergistic toxicity and abrogated the changes in gene expression observed in cells treated with DOX alone. CONCLUSIONS: Treatment with IFN-alpha and DOX should be further explored as a therapeutic strategy for androgen-insensitive prostate cancer.

Induction of ductal and stromal hyperplasia by basic fibroblast growth factor produced by human pancreatic carcinoma.

A histopathology study of 22 pancreatic adenocarcinoma cases revealed that 13 of the patients presented with hyperplastic lesions (atypical and non-atypical hyperplasia, mucous cell hypertrophy, focal epithelial hyperplasia, and ductal papillary hyperplasia), and 9 exhibited fibrosis adjacent to the carcinoma. All lesions expressed high levels of epidermal growth factor receptor (EGF-R) (p<0.0001 and p=0.0008, respectively) as compared with normal ductal epithelium. Non-atypical and atypical hyperplastic lesions also had a higher proliferating cell nuclear antigen (PCNA) labeling index (p<0.001 and p=0.0008, respectively) than normal ductal epithelium. A gradient in PCNA+ nuclei was found in acinar cells adjacent to the tumors. In 16 cases with marked fibrosis, we observed a significant increase of PCNA+ nuclei in stromal fibroblasts (p=0.0041) and significant upregulation of basic fibroblast growth factor (bFGF) mRNA expression in adjacent tumor cells (p=0.0213). These data suggest that the production of bFGF by pancreatic cancer cells induces ductal and stromal hyperplasia of the pancreas.

Optimization for the blockade of epidermal growth factor receptor signaling for therapy of human pancreatic carcinoma.

We determined the optimal administration schedule of a novel epidermal growth factor receptor (EGFR) protein tyrosine kinase inhibitor (PKI), PKI 166 (4-(R)-phenethylamino-6-(hydroxyl)phenyl-7H-pyrrolo[2.3-d]-pyrimidine), alone or in combination with gemcitabine (administered i.p.) for therapy of L3.6pl human pancreatic carcinoma growing in the pancreas of nude mice. Seven days after orthotopic implantation of L3.6pl cells, the mice received daily oral doses of PKI 166. PKI 166 therapy significantly inhibited phosphorylation of the EGFR without affecting EGFR expression. EGFR phosphorylation was restored 72 h after cessation of therapy. Seven days after orthotopic injection of L3.6pl cells, groups of mice received daily or thrice weekly oral doses of PKI 166 alone or in combination with gemcitabine. Treatment with PKI 166 (daily), PKI 166 (3 times/week), or gemcitabine alone produced a 72%, 69%, or 70% reduction in the volume of pancreatic tumors in mice, respectively. Daily oral PKI 166 or thrice weekly oral PKI 166 in combination with injected gemcitabine produced 97% and 95% decreases in volume of pancreatic cancers and significant inhibition of lymph node and liver metastasis. Daily oral PKI 166 produced a 20% decrease in body weight, whereas treatment 3 times/week did not. Decreased microvessel density, decreased proliferating cell nuclear antigen staining, and increased tumor cell and endothelial cell apoptosis correlated with therapeutic success. Collectively, our results demonstrate that three weekly oral administrations of an EGFR tyrosine kinase inhibitor in combination with gemcitabine are sufficient to significantly inhibit primary and metastatic human pancreatic carcinoma.

Blockade of NF-kappaB activity in human prostate cancer cells is associated with suppression of angiogenesis, invasion, and metastasis.

Since the NF-kappaB/relA transcription factor is constitutively activated in human prostate cancer cells, we determined whether blocking NF-kappaB/relA activity in human prostate cancer cells affected their angiogenesis, growth, and metastasis in an orthotopic nude mouse model. Highly metastatic PC-3M human prostate cancer cells were transfected with a mutated IkappaBalpha (IkappaBalphaM), which blocks NF-kappaB activity. Parental (PC-3M), control vector-transfected (PC-3M-Neo), and IkappaBalphaM-transfected (PC-3M-IkappaBalphaM) cells were injected into the prostate gland of nude mice. PC-3M and PC-3M-Neo cells produced rapidly growing tumors and regional lymph node metastasis, whereas PC-3M-IkappaBalphaM cells produced slow growing tumors with low metastatic potential. NF-kappaB signaling blockade significantly inhibited in vitro and in vivo expression of three major proangiogenic molecules, VEGF, IL-8, and MMP-9, and hence decreased neoplastic angiogenesis. Inhibition of NF-kappaB activity in PC-3M cells also resulted in the downregulation of MMP-9 mRNA and collagenase activity, resulting in decreased invasion through Matrigel. Collectively, these data suggest that blockade of NF-kappaB activity in PC-3M cells inhibits angiogenesis, invasion, and metastasis.

Suppression of angiogenesis and therapy of human colon cancer liver metastasis by systemic administration of interferon-alpha.

The purpose of this study was to determine whether systemic administration of interferon-alpha (IFN-alpha) can inhibit liver metastasis produced in nude mice by human colon cancer cells. KM12L4 (IFN-alpha-sensitive) or KM12L4 IFN(R) (IFN-alpha-resistant) cells were injected into the spleen of nude mice. Seven days later, the mice were treated with subcutaneous (s.c.) injections of IFN-alpha (70,000 units/week) at different dosing schedules (1, 2, or 7 times/week). Significant inhibition of tumor growth, vascularization and expression of basic fibroblast growth factor (bFGF) or matrix metalloproteinase-9 (MMP-9) mRNA and protein occurred in mice given daily injections of IFN-alpha. Kinetic analysis of therapy showed that daily s.c. administrations of 10,000 units of IFN-alpha induced apoptosis in liver metastasis-associated endothelial cells, followed by inhibition of tumor cell division and apoptosis of tumor cells. These data suggest that the antiangiogenic activity of IFN-alpha-2a depends on frequent administration of the optimal biologic dose.

Seed and soil revisited: contribution of the organ microenvironment to cancer metastasis.

The outcome of cancer metastasis depends on multiple interactions (cross-talk) between selected metastatic cells and homeostatic mechanisms unique to some organ microenvironments. The organ microenvironment can influence the biology of cancer growth, angiogenesis, and metastasis in several different ways. Therapy of metastasis, therefore, should be targeted against metastatic tumor cells and the homeostatic factors that are favorable to metastasis, growth, and survival of the metastatic cells.

Expression of interleukin-8 in human metastatic endometrial carcinoma cells and its regulation by inflammatory cytokines.

In the present study, we analyzed the expression of a multifunctional cytokine, interleukin-8 (IL-8), in metastatic endometrial carcinoma cells. Our data demonstrate that human serous papillary endometrial adenocarcinoma (SPEC) and human endometrial adenocarcinoma (HEC) cells expressed steady-state IL-8-specific mRNA transcript and secreted IL-8 protein. The levels of IL-8 mRNA in SPEC-2 cells established from stage IV serous papillary adenocarcinoma were three-fold higher as compared to endometrial adenocarcinoma cells, HEC-1 A, established from stage IA endometrial cancer. Further, we observed higher levels of IL-8 mRNA and protein expression in the metastatic variants of SPEC-2 and HEC-1A cells as compared to the parent cell lines, demonstrating that IL-8 expression was associated with metastatic potential. Further, the treatment of endometrial carcinoma cells with inflammatory cytokines, IL-1beta and tumor necrosis factor-alpha (TNF-alpha), demonstrated that IL-1beta and TNF-alpha induced IL-8 expression in endometrial cancer cells. IL-1beta was a more potent inducer of IL-8 expression than TNF-alpha in our studies. These data demonstrate that constitutive and induced IL-8 expression in endometrial carcinoma cells might be an important regulatory mechanism of tumor growth and metastasis.

Expression of interleukin-8 correlates with angiogenesis, tumorigenicity, and metastasis of human prostate cancer cells implanted orthotopically in nude mice.

We determined whether the expression of interleukin-8 (IL-8) by human prostate cancer cells correlates with induction of angiogenesis, tumorigenicity, and production of metastasis. Low and high IL-8-producing clones were isolated from the heterogeneous PC-3 human prostate cancer cell line. The secretion of IL-8 protein correlated with transcriptional activity and levels of IL-8 mRNA. All PC-3 cells expressed both IL-8 receptors, CXCR1 and CXCR2. The low and high IL-8-producing clones were injected into the prostate of nude mice. Titration studies indicated that PC-3 cells expressing high levels of IL-8 were highly tumorigenic, producing rapidly growing, highly vascularized prostate tumors with and a 100% incidence of lymph node metastasis. Low IL-8-expressing PC-3 cells were less tumorigenic, producing slower growing and less vascularized primary tumors and a significantly lower incidence of metastasis. In situ hybridization (ISH) analysis of the tumors for expression of genes that regulate angiogenesis and metastasis showed that the expression level of IL-8, matrix metalloproteinases, vascular endothelial growth factor (VEGF), and E-cadherin corresponded with microvascular density and biological behavior of the prostate cancers in nude mice. Collectively, the data show that the expression level of IL-8 in human prostate cancer cells is associated with angiogenesis, tumorigenicity, and metastasis.

Regulation of neoplastic angiogenesis.

The progressive growth of neoplasms and the production of metastasis depend on the development of adequate vasculature, i.e., angiogenesis. The extent of angiogenesis is determined by the balance between positive- and negative-regulating molecules that are released by tumor and host cells in the microenvironment. The growth of many neoplasms is associated with the absence of the endogenous inhibitor of angiogenesis, interferon beta (IFN beta). A survey of multiple mouse and human tumors shows a lack of IFN beta associated with extensive angiogenesis. Therapy with IFN alpha or beta either by subcutaneous injection of the protein or by introduction of viral vectors that contain the IFN beta gene inhibit angiogenesis and, hence, progressive tumor growth.

Expression levels of genes that regulate metastasis and angiogenesis correlate with advanced pathological stage of renal cell carcinoma.

We examined the expression levels of a number of metastasis-related genes to determine the relationship of these levels to the development of metastasis in renal cell carcinoma. Gene expression was examined in 46 formalin-fixed, paraffin-embedded, archival specimens of primary organ-confined, clear-cell, renal cell carcinoma from patients who had undergone radical nephrectomy. Twenty samples were from patients who did not have metastasis after a median of 48 months; 26 were from patients with either synchronous or metachronous metastases. Microvessel density was assessed by anti-CD-34 immunohistochemical analysis. The expression levels of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), matrix metalloproteinases (MMP)-2 and -9, and E-cadherin were examined at the periphery of the tumor by a colorimetric in situ mRNA. The expression levels of bFGF, VEGF, IL-8, MMP-2, and MMP-9 were significantly higher in primary renal tumors from patients with either synchronous or metachronous metastases than those who were disease-free at a median of 48 months of follow-up. Multivariate analysis of disease-free survival showed that the ratio of MMP-9 to E-cadherin (P = 0.012) and the expression level of bFGF expression (P = 0.045), were independent predictors for the development of metastases. The expression levels of bFGF, VEGF, and IL-8 did not correlate with microvessel density, which in itself was not a significant predictor of progression (P = 0.21). In summary, expression levels of genes that regulate metastasis angiogenesis can predict the metastatic potential in individual patients with organ-confined clear-cell renal carcinoma.

Inhibition of growth and metastasis of human pancreatic cancer growing in nude mice by PTK 787/ZK222584, an inhibitor of the vascular endothelial growth factor receptor tyrosine kinases.

Since vascular endothelial growth factor (VEGF) plays a major role in tumor angiogenesis, we determined whether blockage of VEGF receptor signaling using a novel tyrosine kinase inhibitor (PTK 787) decreases the growth and metastasis of human pancreatic carcinoma growing orthotopically in nude mice. Human pancreatic L3.6pl cells were injected into the pancreas of nude mice. Seven days later, groups of mice were given daily oral administrations of PTK 787 alone, twice weekly i.p. injections of gemcitabine, or combination therapy. The mice were necropsied when control mice became moribund (day 35). Therapy with PTK 787 alone, gemcitabine alone, or the combination of both agents produced respectively 60%, 70%, and 81% inhibition in the volume of pancreatic cancers. The combination therapy significantly decreased the incidence of lymph node and liver metastasis, leading to a significant increase in survival. Microvessel density (MVD) was significantly decreased in tumors treated with either PTK 787 alone or PTK 787 plus gemcitabine. MVD directly correlated with tumor cell proliferation and inversely correlated with apoptosis of tumor cells and associated endothelial cells. Collectively, our results demonstrate that blockade of VEGF-R signaling may provide an additional approach to the therapy of pancreatic cancer.

Production of experimental malignant pleural effusions is dependent on invasion of the pleura and expression of vascular endothelial growth factor/vascular permeability factor by human lung cancer cells.

We determined the molecular mechanisms that regulate the pathogenesis of malignant pleural effusion (PE) associated with advanced stage of human, non-small-cell lung cancer. Intravenous injection of human PC14 and PC14PE6 (adenocarcinoma) or H226 (squamous cell carcinoma) cells into nude mice yielded numerous lung lesions. PC14 and PC14PE6 lung lesions invaded the pleura and produced PE containing a high level of vascular endothelial growth factor (VEGF)-localized vascular hyperpermeability. Lung lesions produced by H226 cells were confined to the lung parenchyma with no PE. The level of expression of VEGF mRNA and protein by the cell lines directly correlated with extent of PE formation. Transfection of PC14PE6 cells with antisense VEGF165 gene did not inhibit invasion into the pleural space but reduced PE formation. H226 cells transfected with either sense VEGF 165 or sense VEGF 121 genes induced localized vascular hyperpermeability and produced PE only after direct implantation into the thoracic cavity. The production of PE was thus associated with the ability of tumor cells to invade the pleura, a property associated with expression of high levels of urokinase-type plasminogen activator and low levels of TIMP-2. Collectively, the data demonstrate that the production of malignant PE requires tumor cells to invade the pleura and express high levels of VEGF/VPF.

Induction of angiogenesis by hyperplastic colonic mucosa adjacent to colon cancer.

We determined whether hyperplastic mucosa adjacent to colon cancer contributes to neoplastic angiogenesis. Surgical specimens of human colon cancer (40 Dukes' stage B and 34 Dukes' stage C) were analyzed by immunohistochemistry for expression of proliferative and angiogenic molecules. The mucosa adjacent to Dukes' stage C tumors (but not Dukes' stage B tumors) had a higher Ki-67 labeling index and a higher expression of epidermal growth factor receptor and transforming growth factor-alpha than distant mucosa. The expression levels of vascular endothelial growth factor, basic fibroblast growth factor, interleukin-8, and the vascular density in the adjacent mucosa were similar to those in the tumor lesions and significantly higher than those in the distant mucosa. The expression of interferon-beta inversely correlated with the level of pro-angiogenic molecules and the vascular density. The injection of metastatic human colon cancer cells and murine colon cancer cells into the cecal wall of mice induced hyperplastic changes in the adjacent mucosa which expressed higher levels of epidermal growth factor receptor, basic fibroblast growth factor, and vascular endothelial growth factor, and lower levels of interferon-beta than did the control mucosa, which directly correlated with the degree of hyperplasia. These data suggest that metastatic human colon cancer cells can induce hyperplasia in the adjacent mucosa, which in turn produces angiogenic molecules that contribute to neoplastic angiogenesis.

Blockade of nuclear factor-kappaB signaling inhibits angiogenesis and tumorigenicity of human ovarian cancer cells by suppressing expression of vascular endothelial growth factor and interleukin 8.

We determined whether blockade of nuclear factor (NF)-kappaB/relA activity in human ovarian cancer cells can suppress angiogenesis and growth in an orthotopic nude mouse model. The human ovarian cancer cells SKOV3ip.1 and HEY-A8 were transfected with a mutated IkappaBalpha (IkappaBalphaM), ie., resistant to phosphorylation and degradation, and hence blocks NF-kappaB activity. NF-kappaB signaling blockade significantly inhibited in vitro and in vivo expression of two major proangiogenic molecules, vascular endothelial growth factor and interleukin 8, in cultured cells and in cells implanted into the peritoneal cavity of nude mice. The decreased expression of vascular endothelial growth factor and interleukin 8 directly correlated with decreased tumorigenicity, decreased vascularization of lesions, decreased formation of malignant ascites, and prolonged survival of mice. These findings suggest that inhibition of NF-kappaB/relA activity in ovarian cancer cells can suppress angiogenesis and progressive growth.