Hassan Adwan

Osteonectin Influences Growth and Invasion of Pancreatic Cancer Cells

Objective:We sought to examine the expression and functional role of osteonectin in primary and metastatic pancreatic ductal adenocarcinoma (PDAC). Background:The glycoprotein osteonectin plays a vital role in cell–matrix interactions and is involved in various biologic processes. Overexpression of osteonectin is present in malignant tumors and correlates with disease progression and poor prognosis. Methods:Expression of osteonectin was analyzed by quantitative polymerase chain reaction and immunohistochemistry in pancreatic tissues and by enzyme-linked immunosorbent assay in the serum of patients and donors. Recombinant osteonectin and specific antisense oligonucleotides were used to examine the effects of osteonectin on induction of target genes, and on proliferation and invasiveness of pancreatic cancer cells. Results:There was a 31-fold increase in osteonectin mRNA levels in PDAC and a 16-fold increase in chronic pancreatitis as compared with the normal pancreas (P < 0.01). By immunohistochemistry, faint immunoreactivity was detected in the normal pancreas. In contrast, strong staining of the cancer cells was observed in addition to extensive osteonectin immunoreactivity in surrounding fibroblasts and in the extracellular matrix. In metastatic tissues, strong immunoreactivity was observed in fibroblasts and in extracellular matrix surrounding metastatic cancer cells, whereas the signal was absent in most tumor cells. In vitro studies showed that osteonectin was able to inhibit cancer cell growth while promoting invasiveness of pancreatic tumor cells. Conclusion:Osteonectin is markedly overexpressed in pancreatic cancer and has the potential to increase the invasiveness of pancreatic cancer cells.

Characterization of a rat model with site-specific bone metastasis induced by MDA-MB-231 breast cancer cells and its application to the effects of an antibody against bone sialoprotein

Metastasis into the skeleton is a serious complication of certain neoplastic diseases such as breast, prostate and lung cancer, but the reasons for this osteotropism are poorly understood. Our aim was to establish a physiologically relevant animal model that is characterized by osteolytic lesions confined to the hind leg of nude rats. For this purpose, we injected 1×105 MDA‐MB‐231 human breast cancer cells transfected with GFP into the superficial epigastric artery, which is an anastomosing vessel between the femoral and iliac arteries. As assessed with the aid of X‐rays, computed tomography and immunohistochemisty, osteolytic lesions occurred exclusively in the femur, tibia and fibula of the animals. The tumor take rate was 93% in a series of 96 rats and the increase in lesion size was observed up to 110 days after tumor cell inoculation. When applying this animal model to the effects of an antibody against bone sialoprotein (BSP), a significantly reduced osteolytic lesion size was observed after preincubation of cells (2 hr, 600 μg/ml anti‐BSP) prior to intra‐arterial tumor cell injection resulting in 19 T/C% at day 60 after tumor implantation (p < 0.05). In addition, the osteolytic lesion size was also significantly reduced after s.c. treatment of the animals with the antibody (20 mg/kg anti‐BSPx3 within 5 days after tumor implantation), resulting in 30 T/C% at day 60 after tumor cell implantation (p < 0.05). In conclusion, the novel rat model for site‐specific osteolytic lesions provides in vivo evidence that preincubation of MDA‐MB‐231GFP cells and treatment of rats after tumor implantation with an antibody against BSP significantly reduces the size of lytic lesions in bone.

Downregulation of osteopontin and bone sialoprotein II is related to reduced colony formation and metastasis formation of MDA-MB-231 human breast cancer cells

Osteopontin (OPN), bone sialoprotein (BSPII), and osteonectin (ON) belong to a family of glycoproteins, which have been linked to cancer metastasis and progression. Here, we report on the selection of antisense oligonucleotides (ASOs), which are effective in reducing their protein levels. In human MDA-MB-231 breast cancer cells, the maximum inhibition of protein expression ranged from 84% (OPN) to 75% (BSPII) and 70% (ON). Erucylphospho-NNN-trimethylpropanolamine (ErPC3) was used as positive control and combination partner. Exposure to ErPC3 inhibited colony formation of MDA-MB-231 cells by 11% (10 μM), 45% (14 μM) and 78% (20 μM). The clonogenicity of breast cancer cells was reduced by 15%, 11%, 8% (5 μM), 39%, 19%, 14% (10 μM) and 46%, 39%, 21% (20 μM) in response to ASO-OPN-04, ASO-BSPII-06 and ASO-ON-03, respectively. Combination of ErPC3 with the ASOs caused additive combination effects. Pre-exposure to the ASOs, but not to the NSO, inhibited formation of osteolytic metastasis in three of four (ASO-OPN-04, P<0.03) and two of four (ASO-BSPII-06) nude rats, and reduced metastasis lesions significantly (T/C%=4.3 and 9.1, P=0.05, respectively). We conclude that downregulation of OPN and BSPII reduces colony formation of MDA-MB-231 cells and formation of osteolytic metastasis in nude rats.

Osteopontin influences the invasiveness of pancreatic cancer cells and is increased in neoplastic and inflammatory conditions

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies, with an overall 5-year survival rate of less then 5%. Invasive tumour growth and early metastasis are two important reasons for this dismal prognosis. Osteopontin (OPN) is a secretory protein with a variety of functions, for example in cell adhesion and migration, inflammatory reaction and apoptosis. In this study the functional role of OPN in human pancreatic cancer and its potential use as a disease marker were analyzed. By real time quantitative PCR, there was a 2.2-fold and 1.6-fold increase of OPN mRNA in pancreatic cancers (n=23) and chronic pancreatitis samples (n=22), respectively, compared to normal pancreatic tissues (n=20). Immunohistochemical analysis demonstrated OPN staining in 60% of the primary pancreatic tumours and in 72% of the lymph node and liver metastases. ELISA analysis of serum samples obtained from pancreatic cancer patients (n=70), chronic pancreatitis patients (n=12), and healthy donors (n=20) showed a 1.6-fold increase in OPN serum levels in patients with tumours and a 1.9–fold increase in patients with chronic pancreatitis. Recombinant human OPN significantly increased the invasiveness of pancreatic cancer cells, without having any impact on cell proliferation. In addition, down-regulation of OPN by specific siRNA molecules decreased pancreatic cancer cell invasion. In conclusion, OPN serum levels in pancreatic cancer and chronic pancreatitis patients are not significantly different, thereby restricting its role as a prognostic or follow-up marker. Our results do suggest, however, that blockade of OPN might be useful as a therapeutic approach to inhibit invasion and metastasis of pancreatic cancer cells.

Sustained delivery and efficacy of polymeric nanoparticles containing osteopontin and bone sialoprotein antisenses in rats with breast cancer bone metastasis

Poor prognosis in mammary carcinoma is associated with a certain expression profile of a defined set of genes including osteopontin and bone sialoprotein. Efficient and specific delivery of antisenses (AS) and a protection of the sequences from degradation are the crucial conditions for AS therapeutic efficiency. We hypothesized that effective and safe AS delivery direceted against these genes could be achieved by polymeric nanoparticles (NP) fabricated from a biocompatible polymer. Due to their nano‐size range and small negative charge, AS‐NP can overcome the absorption barrier offering increased resistance to nuclease degradation, sustained duration of AS administration, and consequently, prolonged antisense action. The ASs designed against OPN and BSP‐II were successfully encapsulated in NP composed of the biodegradable and biocompatible polylactide‐co‐glycolide polymer (PLGA), exhibiting sustained release and stability of the ASs. The therapeutic efficacy of the AS‐NP delivery system was examined in vitro, and in a breast cancer bone metastasis animal model of MDA‐MB‐231 human breast cancer cells in nude rats. Treatment with OPN‐AS or BSP‐AS loaded NP in comparison with osmotic mini‐pumps (locoregional injection and SC implants, respectively) resulted in a significant decrease in both, tumor bone metastasis incidence and in the size of the lesions in rats with metastases. Despite its smaller dose, AS‐NP exhibited a better therapeutic efficacy than osmotic mini‐pumps in terms of lesion ratio at later time periods (8–12 weeks). It may be concluded that AS delivery by NP is a promising therapeutic modality providing stability of the encapsulated AS and a sustained release.

The insulin-like growth factor binding proteins 3 and 7 are associated with colorectal cancer and liver metastasis.

Over the last few decades, a great deal of attention has been directed to the IGF system for its vital role in regulating cell and tissue survival, growth and differentiation. The insulin-like growth factor binding proteins (IGFBPs), a main constituent of this system, have been implicated in the tumorigenesis of colorectal cancer (CRC). In this study, we intended to shed more light on two essential members; IGFBP3 as representative for the six main IGFBPs and IGFBP7 to represent their related proteins (IGFBP-rps). Our experiments on silencing IGFBP3 or IGFBP7 in the two human CRC cell lines SW480, Caco2, and in the rat CRC cell line CC531 show reduced proliferation, colony formation, and for IGFBP3, also reduced migration. The expression of both genes in 68 human CRC samples was higher in UICC stages II and III than in stages I and IV. Additionally, IGFBP3 was negatively correlated with age (p = 0.05) and positively related to IGFBP7 expression (p = 0.0001). Further, in a liver metastasis experiment, the expression of both genes was drastically increased in response to early metastatic growth in vivo. Since these high levels returned gradually to normal thereafter, it could be assumed that the up-regulation of IGFBPs is vital during the process of homing into the liver and early metastatic dissemination. Our results indicate that IGFBP3 and 7 cannot be simply considered as tumor suppressors but have additional properties, which become evident only during cancer progression and metastasis formation.

Osteopontin but not osteonectin favors the metastatic growth of pancreatic cancer cell lines.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer- related death in Western countries and among the malignancies with the worst prognosis. Osteonectin and osteopontin, two proteins of the extracellular matrix, have been found to be up-regulated in PDAC. In the present study the expression of osteopontin mRNA as determined in a panel of 14 human pancreatic cancer cell lines was significantly related to the growth these cell lines in the liver of nude rats (p=0.001) whereas osteonectin showed a trend of being negatively related to pancreatic cancer cell growth in vivo (p=0.10). In an in vitro co-culture model of human Suit2-007 and rat AsML PDAC cells with rat hepatocytes, a clearly increased expression of OPN mRNA was found in the tumor cells. In addition both, down-regulation of osteopontin with specific antisense oligonucleotides and treatment with exogenous rh-osteonectin were associated with reduced cell proliferation. In accordance with the latter finding down-regulation of osteonectin was coupled with increased proliferation. This evidence supports a protumorigenic role of osteopontin and points to an anti-tumorigenic role of osteonectin in PDAC.

Sequential biphasic changes in claudin1 and claudin4 expression are correlated to colorectal cancer progression and liver metastasis

Terminal progression of colorectal cancer (CRC) culminates in liver metastasis. To identify genes that are involved in the metastatic phenotype, cDNA microarrays were used to analyse mRNA expression profiles of colorectal carcinoma (CC)531 rat colon adenocarcinoma cells for changes related to their homing into the liver. Briefly, CC531 cells were intraportally implanted into the liver of Wag‐Rij rats and re‐isolated after 3, 6, 9, 14 and 21 days. Compared to control CC531 cells, claudin1 and claudin4 were among the ≥8‐fold initially down‐regulated genes. The co‐culture of tumour cells with isolated rat hepatocytes and Kupffer cells did not induce down‐regulation of either claudin1 or 4. When the environment effective on circulating tumour cells was simulated by cell culture conditions favouring their adhesion, only claudin4 showed augmented expression. Knockdown of claudin1 and claudin4 mediated by small interfering RNA caused significantly increased migration and decreased clonogenic growth of tumour cells (P < 0.05), but had no effect on their proliferation. These experimental results were paralleled by increased claudin1 and claudin4 expression in human CRC samples in Union for International Cancer Control (UICC) stages I–III, as evaluated by real‐time PCR. Increased claudin4 levels were correlated with significantly reduced overall survival (log‐rank test, P= 0.018). Further, significantly (P < 0.05) reduced expression of claudin1 and claudin4 was observed in stage IV and liver metastasis by immunohistochemistry. In conclusion, sequential biphasic changes in claudin1 and claudin4 expression occur during the homing of rat CC531 CRC cells to the liver. This modulation is reflected by significant changes in claudin expression in human primary and metastatic CRC.

Riproximin is a recently discovered type II ribosome inactivating protein with potential for treating cancer.

The development of new anticancer drugs is a salient problem and the traditional use of plants is a potentially rich source of information for detecting new molecules with antineoplastic activity. Riproximin is a recently detected cytotoxic type II ribosome inactivating protein with high selectivity for certain tumor cell lines. Its activity was recognized as the main component in a plant powder used by African healers for treating cancer. By ribulose bisphosphate carboxylase gene sequencing analysis, the powder was identified to be derived from the plant Ximenia americana. The cDNA sequence of riproximin was identified, the protein was modeled to contain one A- and a B-chain, respectively, and a reliable purification procedure from kernels of X. americana was established. Riproximin displays high but differential antiproliferative activity in a panel of human and rodent cancer cell lines, with concentrations inhibiting cell proliferation by 50% (IC50 values) that diverge by a factor of 100. Consistent antineoplastic activity was detected in colorectal and pancreatic cancer liver metastasis models in rats. The cytotoxic mechanism of action was determined to be based on cellular uptake of riproximin followed by its A-chain prompted depurination of the 28S ribosomal RNA and induction of unfolded protein response. Riproximins specificity depended on its B-chain connected binding to cell surface glycans, the presence of which is crucial for subsequent internalization into cells and cytotoxicity. These N- and O-glycans include bi- and tri-antennary NA structures (NA2/NA3) as well as Tn3 structures (clustered Tn antigen). Riproximin was found to crosslink proteins with N- and O-glycan structure, thus indicating both types of binding sites on its B chain. Due to this crosslinking ability, riproximin is expected to show prominent cytotoxicity towards cells expressing both, NA2/NA3 and clustered Tn structures. Apart from the properties of riproximin, the plant X. americana has been known for some medical uses in traditional African medicine, including various types of infections.

Biodistribution of antisense nanoparticles in mammary carcinoma rat model.

Efficient and specific delivery of antisenses (ASs) and protection of the sequences from degradation are critical factors for effective therapy. Sustained release nanoparticles (NP) offer increased resistance to nuclease degradation, increased amounts of AS uptake, and the possibility of control in dosing and sustained duration of AS administration. The biodegradable and biocompatible poly(D,L-lactic-co-glycolic acid) copolymer (PLGA) was utilized to encapsulate AS directed against osteopontin (OPN), which is a promising therapeutic target in mammary carcinoma. Whole body biodistribution of OPN AS NP was evaluated in comparison to naked AS, in intact and mammary carcinoma metastasis model bearing rats. Naked and NP encapsulated AS exhibited different biodistribution profiles. AS NP, in contrast to naked AS, tended to accumulate mostly in the spleen, liver, and at the tumor inoculation site. Drug levels in intact organs were negligible. The elimination of naked AS was faster, due to rapid degradation of the unprotected sequence. It is concluded that AS NP protect the AS from degradation, provide efficient AS delivery to the tumor tissue, and minimize AS accumulation in intact organs due to the AS sustained release profile as well as the favorable NP physicochemical properties.