Purpose: Our aims were to establish and characterize primary human hepatocellular carcinoma xenografts. They were used to screen new drugs and improve our current treatment regimens used in hepatocellular carcinoma. Experimental Design: Primary hepatocellular carcinomas were used to create the xenografts. Western blotting was used to determine the changes in proteins in these xenografts before and after therapies. Apoptotic and cell proliferation were analyzed by immunohistochemistry. Results: Seven lines of xenografts were established from primary human hepatocellular carcinomas. Lines 4-1318, 2-1318, 2006, and 26-1004 grew rapidly in severe combined immunodeficient (SCID) mice and doubled its volume every 48 to 72 hours. Series 5-1318 (5-1318, 30-1004, and 29-1104) grew relatively slowly in SCID mice and required ∼6 to 10 days to double its tumor volume. Western blot analysis revealed that the growth rate of these xenografts was associated with abnormal expression of proteins associated with the cell cycle, signaling pathways, and tumor suppressor genes. Although hepatocellular carcinoma xenografts expressed the receptors for androgens, estrogens, and progesterone, their growth rate was not affected by either castration or sex steroid hormone supplementation. Cisplatin, oxaliplatin, vitamin D analogue EB1089, and Iressa had no effects on the growth rate in SCID mice. Although 5-fluorouracil exerted mild growth inhibition of these xenografts, i.p. delivery of 2-chloroethyl-3-sarcosinamide-1-nitrosourea (SarCNU) or doxorubicin resulted in a significant growth inhibition. Doxorubicin-induced growth suppression was associated with elevation of p53 and p21Cip1/Waf1. In addition to up-regulation of p53 and p21Cip1/Waf1, SarCNU also increased the levels of phosphorylated cdc-2 at Tyr15. Conclusion: Hepatocellular carcinoma xenografts are powerful tools for screening drugs and SarCNU may be useful in the treatment of this fatal disease.

Hepatocellular carcinoma (HCC) is a common malignancy in Asia and Africa. We previously reported that overexpression of extracellular signal-regulated kinase (ERK) kinase 1/2 (MEK1/2) and ERK1/2 was detected in HCC, and that their activation was required for liver cancer cell proliferation and survival. In the present study, we determined the efficacy of a specific MEK1/2 inhibitor AZD6244 (ARRAY-142886) in treatment of HCC. Treatment of primary HCC cells with AZD6244 led to growth inhibition, elevation of the cleavage of caspase-3 and caspase-7, and cleaved poly(ADP)ribose polymerase, but inhibition of ERK1/2 and p90RSK phosphorylation. Studying the protein expression profile of seven HCC xenografts revealed that their growth rate was positively correlated with the levels of phosphorylated MEK. AZD6244, when given p.o. to mice bearing these xenografts, resulted in a dose-dependent inhibition of tumor growth. AZD6244-induced growth suppression was associated with inactivation of ERK1/2 and p90RSK, and up-regulation of activated caspase-3 and caspase-7, and cleaved poly(ADP)ribose polymerase. Our data suggest that the MEK-ERK pathway plays an important role in the growth and survival of liver cancer cells and that the HCC xenograft models are excellent tools for screening preclinical drugs.

MCF-7 human breast cancer cells are commonly used to model tissues responsive to estrogens and antiestrogens. We examined the effects of estradiol and the antiestrogen ICI 182780 on MCF-7 cell proliferation and insulin-like growth factor binding protein 3 (IGFBP-3) gene expression. ICI 182780-induced growth inhibition was associated with increased transcription of the IG-FBP-3 gene, increased IGFBP-3 mRNA abundance, and increased IGFBP-3 protein accumulation in the conditioned medium. The growth stimulatory effect of estradiol was associated with opposite effects, and the correlation between cellular proliferation and IGFBP-3 mRNA abundance was strong (r ‫؍‬ ؊0.91). Recombinant IGFBP-3 inhibited basal and estradiol-stimulated MCF-7 cell proliferation, and an IGFBP-3 antisense oligodeoxynucleotide abolished antiestrogen-induced growth inhibition. These results provide evidence for an estradiol and antiestrogen-regulated IGFBP-3 growth inhibitory autocrine pathway in MCF-7 cells. Insulin-like growth factors I and II (IGF-I1 and IGF-II) are potent mitogens and inhibitors of apoptosis for many normal and neoplastic cell types, including normal and transformed breast epithelial cells (1, 2). Both IGF-I and IGF-II bind with high affinity to specific IGF-binding proteins (IGFBPs), which modulate their bioactivity. At least six IGFBPs have been described (3,4). IGFBP-3 acts as a growth inhibitor in many (but not all (5)) experimental systems (reviewed in Ref. 6). Examples of data consistent with a growth inhibitory role for IGFBP-3 include the growth inhibition associated with IG-FBP-3 gene transfection (6), the increased IGFBP-3 accumulation associated with senescence-related reduction of cellular proliferation (7), the increase in IGFBP-3 production associated with retinoid-induced growth inhibition (8), and the decrease in IGFBP-3 production associated with epidermal growth factorstimulated proliferation (9). Early studies attributed the growth inhibitory action of IGFBP-3 to the reduction of IGF-I and/or IGF-II bioactivity resulting from competition for somatomedins between IGFBP-3 and the type I IGF receptor (10). However, there is recent evidence that IGFBP-3 also has growth inhibitory activity that is independent of its IGF binding properties (11)(12)(13)(14).Antiestrogens are widely used in breast cancer treatment, and it has been proposed that the inhibitory effect of these compounds on IGF-I expression contributes to their antiproliferative activity (15, 16) (reviewed in Ref. 17). Antiestrogens have significant trophic effects on the uterus (18), and there is a negative correlation between uterine weight and uterine IG-FBP-3 expression; the positive uterotrophic actions of both estradiol and the partial estrogen receptor antagonist tamoxifen are associated with suppression of uterine IGFBP-3 expression, while the pure antiestrogen ICI 182,780 (19) causes uterine involution and markedly enhances uterine IGFBP-3 expression (20). We undertook the present study to examine the possibility that the anti-proliferativ...

Introduction Hepatocellular carcinoma (HCC) is the fifth most common primary neoplasm worldwide, with approximately 660,000 deaths worldwide annually [1,2]. Recurrence, metastasis and the development of new primary tumours are the most common causes of mortality for patients with HCC [3,4] Tyr1021, phospho-eIF4E Ser209, phospho-c-Raf Ser259, c-Raf, Mcl-1, Bcl-2, Bcl-x Abstract Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide. Vascular endothelial growth factor, platelet derived growth factor and the Raf/mitogen-activated protein kinase/extracellular signal regulated kinase (Raf/MEK/ERK) signalling pathway regulates the growth, neovascularization, invasiveness and metastatic potential of HCC. In this study, we investigated the in vivo antitumour activity and mechanisms of action of sorafenib tosylate on four patient-derived HCC xenografts. Sorafenib dosed at 50 mg/kg and 100 mg/kg inhibited tumour growth by 85% and 96%, respectively. Sorafenib-induced growth suppression and apoptosis were associated with inhibition of angiogenesis, down-regulation of phospho-platelet-derived growth factor receptor ␤

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death in the world. The multikinase inhibitor sorafenib only demonstrated marginal improvement in overall survival for advanced disease prompted the search for alternative treatment options. Human mesenchymal stem cells (MSCs) have the ability to home to tumor cells. However, its functional roles on the tumor microenvironment remain controversial. Herein, we showed that conditioned media derived from human fetal MSC (CM-hfMSCs) expressed high level of the insulin growth factor binding proteins IGFBPs and can sequester free insulin-like growth factors (IGFs) to inhibit HCC cell proliferation. The inhibitory effect of IGFBPs on IGF signaling was further evident from the reduction of activated IGF-1R and PI3K/Akt, leading eventually to the induction of cell cycle arrest. We also demonstrated that CM-hfMSCs could enhance the therapeutic efficacy of sorafenib and sunitinib. To the best of our knowledge, this is the first report to show that CM-hfMSCs has a tumor-specific, antiproliferative effect that is not observed with normal human hepatocyte cells and patient-derived matched normal tissues. Our results thus suggest that CM-hfMSCs can provide a useful tool to design alternative/adjuvant treatment strategies for HCC, especially in related function to potentiate the effects of chemotherapeutic drugs.

tects cells from apoptosis, and have implicated the serInsulin-like growth factors (IGFs) are known to ine-threonine kinase Akt as a key downstream regulahave potent antiapoptotic activity. The antiestrogen tor of the antiapoptotic effect of IGF-IR agonists (7). ICI 182,780 (ICI) is a potent inhibitor of MCF7 human However, little attention has been given to date to the breast cancer cell growth and has recently been re-role of insulin-like growth factor binding proteins ported to act as an antiproliferative agent in part via (IGFBPs) (8,9) in regulating apoptosis. ing a cell death ELISA (Boehringer Mannheim) which measures cytoplasmic histone-bound DNA generated during apoptotic DNA fragmentation and not free histone or DNA that could be released during nonapoptotic cell death. This method has been shown to be reliable for quantitation of apoptotic cell death in MCF7 cells (13,14). MCF7Apoptosis is a physiological phenomenon involved in cells plated at a density of 25,000 cells/well and treated for 72 hours morphogenesis and tissue renewal (1). Transformed exactly as described for growth experiments were harvested by scraping in cold phosphate buffered saline. Parallel plates treated cells have been shown to have defects in activation of identically were used for thymidine incorporation and for determinathe apoptotic pathway, suggesting that derangements tion of cell number. Cytoplasmic cell extracts were prepared acof apoptosis are involved in the pathophysiology of can-cording to the manufacturer's protocol and were equalized on the cer. Many antineoplastic compounds induce tumor re-basis of cell number. Samples from triplicate wells were run in duplicate on the ELISA. gression through their ability to activate apoptotic pathways (2,3). Recent studies ((4,5)

Keloids are proliferative dermal growths representing a pathological wound-healing response. We report high proliferation rates in normal (NF) and keloid-derived fibroblasts (KF) cocultured with keloid-derived keratinocytes (KK). IGF binding protein (IGFBP)-3 mRNA and secreted IGFBP-3 in conditioned media were increased in NF cocultured with KK compared with NF but markedly reduced in KF cocultured with KK or normal keratinocytes (NK). IGFBP-2 and IGFBP-4 mRNA levels were elevated, whereas IGFBP-5 mRNA was decreased in KF cocultured with KK or NK. Significant increases in IGFBP-2 and -4 mRNA in KF cocultured with KK did not correlate with protein secretion. Downstream IGF signaling cascade components, phospho-Raf, phospho-MEK1/2, phospho-MAPK, PI-3 kinase, phospho-Akt, and phospho-Elk-1, were elevated in KF cocultured with KK. Addition of recombinant human IGFBP-3 or antibodies against IGF-I or IGF-IR significantly inhibited proliferation of KF. The bioavailability of IGF-I may be related to the levels of IGFBP-3 produced, which in turn influences KF proliferation, suggesting that modulation of IGF-I, IGF-IR, and IGFBP-3, individually or in combination, may represent novel approaches to the treatment of keloids.

We previously demonstrated the growth inhibitory property of OKL38 and its possible roles in mammary carcinogenesis. To further understand the regulation and roles of OKL38 in tumorigenesis we proceeded to clone and characterize the human OKL38 gene and three of its variants with transcripts of 1.9, 2.2, and 2.4 kb. The human OKL38 gene spans ϳ18 kb and contains 8 exons and 7 introns with exon size ranging from 92 to 1270 bp. RT-PCR and sequence analysis suggest that different transcripts were arrived through differential promoter usage and alternate splicing. Multiple Tissue Expression array (MTE) and Multiple Tissue Northern blot (MTN) indicated that OKL38 was ubiquitously expressed in all tissues with high expression in liver, kidney, and testis. The cancer profiling array (CPA) of paired normal/tumor cDNA showed that OKL38 mRNA was down-regulated in 70% (14 of 20) of kidney tumors. Western analysis revealed that the OKL38 protein was undetectable in 78% (7 of 9 pairs) of kidney tumor tissues. Immunohistological analysis showed that 64% (14 of 22) of kidney tumors were either lost or underexpressed OKL38 protein compared with the adjacent normal tissue. A transfection study using OKL38-eGFP recombinant construct showed that overexpression of the 52 kDa OKL38 protein in A498 cells resulted in growth inhibition and cell death. This study demonstrates the complex genomic structure of the OKL38 gene and its growth inhibitory and cytotoxic properties. Our data suggest the potential use of OKL38 in diagnosis, prognosis, and/or treatment of kidney cancer.