The novel cytotoxic polybisphosphonate osteodex decreases bone resorption by enhancing cell death of mature osteoclasts without affecting osteoclastogenesis of RANKL-stimulated mouse bone marrow macrophages.

It has previously been demonstrated that the polybisphosphonate osteodex (ODX) inhibits bone resorption in organ-cultured mouse calvarial bone. In this study, we further investigate the effects by ODX on osteoclast differentiation, formation, and function in several different bone organ and cell cultures. Zoledronic acid (ZOL) was used for comparison. In retinoid-stimulated mouse calvarial organ cultures, ODX and ZOL significantly reduced the numbers of periosteal osteoclasts without affecting Tnfsf11 or Tnfrsf11b mRNA expression. ODX and ZOL also drastically reduced the numbers of osteoclasts in cell cultures isolated from the calvarial bone and in vitamin D3-stimulated mouse crude bone marrow cell cultures. These data suggest that ODX can inhibit osteoclast formation by inhibiting the differentiation of osteoclast progenitor cells or by directly targeting mature osteoclasts. We therefore assessed if osteoclast formation in purified bone marrow macrophage cultures stimulated by RANKL was inhibited by ODX and ZOL and found that the initial formation of mature osteoclasts was not affected, but that the bisphosphonates enhanced cell death of mature osteoclasts. In agreement with these findings, ODX and ZOL did not affect the mRNA expression of the osteoclastic genes Acp5 and Ctsk and the osteoclastogenic transcription factor Nfatc1. When bone marrow macrophages were incubated on bone slices, ODX and ZOL inhibited RANKL-stimulated bone resorption. In conclusion, ODX does not inhibit osteoclast formation but inhibits osteoclastic bone resorption by decreasing osteoclast numbers through enhanced cell death of mature osteoclasts.

Cytotoxicity of poly-guanidine in medulloblastoma cell lines.

Medulloblastoma (MB) is the most common pediatric brain tumor. The therapy frequently causes serious side effects, and new selective therapies are needed. MB expresses hyper sialylation, a possible target for selective therapy. The cytotoxic efficacy of a poly guanidine conjugate (GuaDex) incubated with medulloblastoma cell cultures (DAOY and MB-LU-181) was investigated. The cells were incubated with 0.05-8 µM GuaDex from 15 min to 72 h. A fluorometric cytotoxicity assay (FMCA) measured the cytotoxicity. Labeled GuaDex was used to study tumor cell interaction. FITC-label Sambucus nigra confirmed high expression of sialic acid (Sia). Immunofluorescence microscopy was used to visualize the cell F-actin and microtubules. The cell interactions were studied by confocal and fluorescence microscopy. Annexin-V assay was used to detect apoptosis. Cell cycle analysis was done by DNA content determination. A wound-healing migration assay determined the effects on the migratory ability of DAOY cells after GuaDex treatment. IC50 for GuaDex was 223.4 -281.1 nM. FMCA showed potent growth inhibition on DAOY and MB-LU-181 cells at 5 uM GuaDex after 4 h of incubation. GuaDex treatment induced G2/M phase cell cycle arrest. S. nigra FITC-label lectin confirmed high expression of Sia on DAOY medulloblastoma cells. The GuaDex treatment polymerized the cytoskeleton (actin filaments and microtubules) and bound to DNA, inducing condensation. The Annexin V assay results were negative. Cell migration was inhibited at 0.5 µM GuaDex concentration after 24 h of incubation. GuaDex showed potent cytotoxicity and invasion-inhibitory effects on medulloblastoma cells at low micromolar concentrations. GuaDex efficacy was significant and warrants further studies.

Poly-guanidine shows high cytotoxicity in glioma cell cultures and glioma stem cells.

Glioblastoma multiforme (GBM) is a malignant CNS tumor with a poor prognosis. GBM shows aberrant glycosylation with hypersialylation. This property is a potential target for therapy. This study investigates the growth inhibitory efficacy of poly-guanidine (GuaDex), with an affinity for sialic acid (Sia). Glioma cell cultures and patient-derived glioma cell lines (PDGCLs) expressing Prominin-1 (CD133) were used. Human fibroblasts and astrocyte-derived cells were used as controls. Temozolomide (standard GBM drug, TMZ) and DMSO were used as a comparison. GuaDex at 1-10 µM concentrations, were incubated for 3.5-72 h and with PDGCLs cells for 6-24 h. The cytotoxicity was estimated with a fluorometric cytotoxicity assay (FMCA). Fluorescence-labelled GuaDex was used to study the cell interactions. Sia expression was confirmed with a fluorescence labelled Sia binding lectin. Expression of glial fibrillary acidic protein was determined. GuaDex induction of growth inhibition was fast, showing after less than 5 min incubation while the control cells were not affected even after 50 min incubation. The growth inhibitory effect on PDGCLs spheroids was persistent still showing after 4 weeks post-treatment. The growth inhibition of GuaDex was induced at low µM concentrations while TMZ induced only a slight inhibition at mM concentrations. GuaDex efficacy appears significant and warrants further studies.

Synthesis and Binding of a Novel PSMA-specific Conjugate.

BACKGROUND/AIM: Prostate-specific membrane antigen (PSMA) is emerging as a target for treatment of castration-resistant prostate cancer (CRPC) while its up-regulated in the majority of CRPC tumors. The most common approach is targeted radionuclide therapy. MATERIALS AND METHODS: The PSMA binding pharmacophore Glu-Urea-Lysine (GUL) and lysine were conjugated to oxidized dextran with reductive amination and subsequently labelled with fluorosceinisothiocyanate (FITC). Three prostate cancer cell lines were used for binding studies, 22Rv1 (PSMA positive), DU145 (PSMA negative) and PC3 (PSMA negative). Binding images were obtained by fluorescence microscopy. RESULTS: PDC binding was recorded on the 22Rv1 cell line while the negative cell lines showed no or slight background binding. PDC binding could be inhibited by pre-incubation with a molar excess of unlabelled PDC. CONCLUSION: This is a novel template for PSMA targeted CRPC therapy, either using cytostatics or radionuclides.

Bone-targeted Novel Cytotoxic Polybisphosphonate Conjugate in Castration-resistant Prostate Cancer: A Multicenter Phase 1 Study.

BACKGROUND: Osteodex (ODX) is a cytotoxic bone-targeting polybisphosphonate, intended for treatment of bone metastasis from castration-resistant prostate cancer (CRPC). The primary objective of this study was to describe the tolerability and toxicity of such treatment by defining its maximum tolerated dose (MTD) and dose-limiting toxicity (DLT). PATIENTS AND METHODS: Twenty-eight patients with castration-resistant prostate cancer and confirmed bone metastasis were assigned to seven infusions of ODX every third week, divided in seven ascending dose cohorts. RESULTS: No DLT's were observed and as pre-specified, the highest dose administered was defined as MTD. In total, 206 adverse events (AE) were recorded and 13,6% were classified as treatment-related, while none were serious or severe (SAE). No cumulative toxicity and no renal toxicity were recorded. CONCLUSION: ODX was well tolerated, with few and mild side-effects and with apparent treatment efficacy in the highest dose cohort. Further clinical development is currently in progress.

Proteomic analysis of soft tissue tumor implants treated with a novel polybisphosphonate.

BACKGROUND: Osteodex is a novel bi-functional macromolecular polybisphosphonate developed for treatment of bone metastases in prostate and breast cancer. High efficacy of osteodex has been demonstrated both in vitro and in vivo. The present study investigates whether osteodex is also efficacious on soft tissue tumor lesions. MATERIALS AND METHODS: Twelve female nude mice were injected with MDA-MB-231 cells orthotopically. Osteodex was administered i.v. at 2.5 mg/kg, once per week for five weeks. Tumor volumes were measured during the treatment period, the animals were sacrificed, and samples collected for proteomic analysis. RESULTS: The non-treated mice developed multiple tumors greater than 4 cm with pronounced ulceration, while the treated mice had tumors smaller than 1 cm, without ulceration. While general condition of treated mice was good, non-treated animals were in poor condition. Sixteen out of 300 identified proteins were differentially expressed, with statistically significant expression changes of more than two-fold differences between treated and non-treated groups. These proteins were identified using non-gel based nano-liquid chromatography coupled with a Synapt G2 instrument. CONCLUSION: We conclude that osteodex showed significant treatment efficacy on soft tissue tumor implants. The study provides a global view of changes in protein expression profiles following osteodex treatment. Some functions of the identified proteins might be used to explain the specific treatment efficacy of osteodex.

Treatment of bone metastasis in prostate cancer: efficacy of a novel polybisphosphonate.

AIM: To investigate the in vivo efficacy of a novel polybisphosphonate (ODX) in the treatment of bone metastasis from prostate cancer. MATERIAL AND METHODS: A rat prostatic carcinoma model was used. Forty-two rats (21 control, 21 treatment) had induction of bone lesions through injection of AT6.1 cells into the distal medullar cavity of long bones (right femur). At day 21 post injection, radiographs were taken and tumor score (severity of lesions, 0-4) and tumor incidence (score >0) were determined. Treatment started at day 23 and lasted until day 49 (four i.v. administrations of ODX during four weeks). RESULTS: ODX reduced the severity of the lesions compared to the control group. Forty-seven percent of the treated rats had regression of their lesions at the study end, including four rats showing disappearance of the lesions i.e. score 0. Osteocondensation at the growth plate was only observed in the treatment group, indicating osteoclast inhibition. CONCLUSION: In spite of a relatively short treatment period with only four administrations, ODX showed significant efficacy (p=0.0023), with inhibition of tumor progression and osteolysis. The results are encouraging, confirming previous in vitro studies. Clinical research is pending on patients with bone metastasis from castration-resistant prostate cancer.

Application of accelerator mass spectrometry to macromolecules: preclinical pharmacokinetic studies on a polybisphosphonate.

Data on the use of accelerator mass spectrometry (AMS) in conjunction with in vivo studies of macromolecular drugs are scarce. The present study shows the versatility of this technique when investigating the pharmacokinetics (PK) of a macromolecular drug candidate, a polybisphosphonate conjugate (ODX). The aforementioned is a polymer (molecular weight ~30 kDa) constituting a carbohydrate backbone with covalently linked ligands (aldendronate and aminoguanidine) and is intended for treatment of osteoporosis and the therapy of bone metastasis from prostate cancer. The conjugate is prepared through partial oxidation of the carbohydrate and sequential coupling of the ligands by reductive amination. (14)C was incorporated in the conjugate by means of coupling a commercially available (14)C-lysine in the conjugation sequence. Fifteen rats were injected intravenously with (14)C-labelled ODX (150 µg, 14 Bq/rat) and blood samples were collected at 1, 2, 4, 6, and 24 h post-injection (3 rats/time point). Liver, spleen and kidney samples were collected at 4 and 24 h post-injection. Blood from each time point (triplicate) were collected for AMS measurement determining the isotopic ratio ((14)C/(12)C) and consequently the drug concentration in blood. ODX showed a transient presence in blood circulation; 93% of the total dose was cleared from the circulation within 1 h. The half-life after 1 h was estimated to be about 3 h; 0.7% of the administered (14)C dose of ODX remained in circulation after 24 h. The major (14)C accumulation was in the liver, the spleen and the kidneys indicating the probable route of metabolism and excretion. This study demonstrates the versatility of AMS for pharmacological in vivo studies of macromolecules. Labelling with (14)C is relatively simple, inexpensive and the method requires minimal radioactivity, eliminating the need for radioprotection precautions in contrast to methods using scintillation counting.

Development of a novel poly bisphosphonate conjugate for treatment of skeletal metastasis and osteoporosis.

Advanced stage prostate and breast cancer frequently metastasize to the skeleton (approximately 75%). An additional complication in these patients, that further affects the bones, is that their hormonal treatment, induces osteoporosis. Bisphosphonates (bpns) are standard drugs against osteoporosis and have been shown to have clinically significant anti-tumor effects. This study describes the development of a new polybisphosphonate conjugate (ODX) with enhanced dual efficacy i.e. with anti-bone resorption and anti-tumor properties. Zoledronic acid (Zometa) was used as a positive control (at equimolar concentrations). Alendronic acid and aminoguanidine were conjugated to oxidized dextran with subsequent reductive amination (on average approximately 8 alendronate and approximately 50 guanidine moieties per conjugate). ODX was tested in a bone resorption assay for its capacity to inhibit bone resorbing osteoclasts (bone organ culture from neonatal mice, 45Ca labelled bone mineral). Tumor cell toxicity was studied on prostate (PC3) and breast cancer (MDA231, MDA453) cell cultures. Two methods were employed, a fluorescent cytotoxicity assay (FMCA) and an apoptosis assay (Annexin V assay). In the bone resorption assay, Zometa and ODX showed very similar potency with 50% osteoclast inhibition at approximately 20 nM and 100% at 0.2 microM. In the FMCA, IC50 for ODX was at approximately 2 microM and 25 microM for Zometa (PC3). In the apoptosis assay, ODX induced approximately 85-97% apoptosis at 10 microM in both cell lines, while Zometa failed to induce any significant apoptosis in any of the cell lines at the tested concentration range (10 nM-10 microM). ODX appears to be a promising drug candidate with high dual efficacy for the treatment of bone metastasis and osteoporosis. It has both potent osteoclast inhibiting properties and enhanced anti-tumor efficacy.

The use of Butea superba (Roxb.) compared to sildenafil for treating erectile dysfunction.

OBJECTIVE: To evaluate the effect of an extract of Butea superba (Roxb.) (BS) compared to sildenafil for treating erectile dysfunction (ED). PATIENTS AND METHODS: An open label study was carried out among 32 men with organic ED to evaluate the response on the International Index of Erectile Function 5 (IIEF-5) to BS, a 'natural health' product (100 mg), compared to 50 mg of sildenafil (a phosphodiesterase-5 inhibitor). After a 1-week wash-out, responders to BS received either 100 mg starch or 100 mg of another batch of BS (double-blind). RESULTS: Of the patients in the BS group, 27 (84%) responded positively, compared with 26 (81%) in the sildenafil group. When assessing the score alone, 12 (38%) had a better score after taking BS, compared to seven (22%) after sildenafil, and eight (25%) had the same score. The results were surprising and could not be repeated in the double-blind part of the study, where no effect of BS was recorded. CONCLUSIONS: A 'natural' health product containing BS was more effective than sildenafil in the first part of the study, but in the second, using another batch of BS, the positive result could not be repeated and no effect was recorded. The conclusion is that the first preparation of BS was most likely blended with a phosphodiesterase-5 inhibitor, later confirmed by the supplier of BS (a natural health products company) after their own analysis.

Comparison of protein expression in two prostate cancer cell-lines, LNCaP and DU145, after treatment with somatostatin.

The mechanisms underlying prostate cancer progression are poorly understood. Proteins responsive to androgens may be involved in the development and progression of prostate cancer and the ultimate failure of androgen-ablation therapy. Therapy with somatostatin (sms) analogues could be a possible therapeutic alternative to chemotherapy in hormone refractory prostate cancer patients. We used two prostate cancer cell-lines, LNCaP (androgen-dependent) and DU145 (androgen-independent), to compare the protein expressions. Both cell lines were treated with sms and its derivative smsdx. Smsdx is a glycosylated poly sms with high stability suitable for clinical use. A comparison study of protein expression was analyzed by means of two-dimensional gel electrophoresis (2DE) followed by mass spectrometric analysis. Marked quantitative differences were observed in the protein expression profiles in sms/smsdx treated LNCaP and DU145 cells compared to the control cells. One third of the detected proteins were differentially expressed (PRDXs, hnRNPs, HSPs, RKIP). Concordance in protein expression patterns was observed between smsdx and sms treated cells with strong agreement between the up- and down-regulation of proteins. Fifty-eight (isoforms of 49 proteins) protein spots were identified and found differentially expressed at 2-fold change between LNCaP and DU145 cells. Thirty-one proteins in LNCaP have higher expressions than in DU145. Twenty-seven proteins in DU145 have higher expressions than in LNCaP. Most of the differentially expressed proteins (2-fold) between LNCaP and DU145 cells were affected by sms/smsdx treatment (1.2- to 2.6-fold change). Sms/smsdx affects the mitochondria of prostate cancer cells in a way that eventually triggers mitochondrial-mediated apoptosis. Regulation of certain proteins (e.g., RKIP, VDACs) by sms/smsdx suggests that sms/smsdx exerts its effects on prostate cancer cells via MAPK pathway and by regulating the activities of phosphotyrosine phosphatases.

Polymer-conjugated guanidine is a potentially useful anti-tumor agent.

Guanidine compounds have important biochemical properties. Aminoguanidine, as an example, is an anti-oxidant, a nitric oxide synthase inhibitor (NOS) which prevents nitric oxide formation, and an inhibitor of advanced glycosylation end products (AGEs). As an anti-oxidant, aminoguanidine may affect the formation of atherosclerotic lesions through protection from LDL oxidation. Inhibition of AGEs could have a preventive effect on the tissue damage caused by diabetes where AGEs are considered to be an important factor. The role of NO in cancer is complex and not fully understood, but it may have influence on growth and progression. In this study, the tumor growth inhibitory effect of conjugated guanidine (i.e. a polyguanidine) was investigated. The effect on tumor cell growth was studied in cultures of prostate, breast, bladder and renal cell cancer, and a fluorometric cytotoxicity assay was performed. Guanidine conjugates were prepared by reacting aminoguanidine or agmatine with periodate oxidized dextran followed by reductive amination. The cytotoxic effect was compared with an anthracycline (adriamycin). The dextran-guanidine conjugates were cytotoxic at low micromolar concentrations, and the dextran-aminoguanidine conjugate (GDC) had the highest efficacy, being more efficient than adriamycin, in all of the tested tumor cell lines. Breast and prostate cancer cells were the most sensitive. At 0.5 microM, GDC killed >95% of the breast cancer cells compared to 25% for Adriamycine. In prostate cancer cells, GDC killed approximately 55% of the cells at 0.1 microM and 100% of the cells at 0.5 microM compared to approximately 22 and approximately 62%, respectively, for adriamycin. Unconjugated aminoguanidine and agmatine did not seem to affect tumor cell growth even at high concentrations (mM). Polymer- conjugated guanidine is a potentially useful template for the construction of therapeutic tumor targeting cytotoxic agents.

Incubation with somatostatin, 5-aza decitabine and trichostatin up-regulates somatostatin receptor expression in prostate cancer cells.

Somatostatin (SMS), binds to its specific receptors (SSTRs) and transduces growth inhibitory, anti-secretory and apoptotic signals. Several human cancers express SSTRs, including prostate cancer, and therefore SMS is of interest for anti-cancer therapy. DNA methylation and histone modifications are involved in normal cell development, gene imprinting and human carcinogenesis. Reversing DNA methylation is an attractive therapeutic possibility, since epigenetic modifications change gene expression without changing the gene function. DNA methylation inhibitors such as 5-aza-2'-deoxycytidine (5'-aza, decitabine) have been used to treat several types of haematological malignancies. Histone deacetylase inhibitors such as trichostatin (TSA), are a new class of 'targeted anti-cancer agents'. TSA and decitabine can induce growth arrest, apoptosis or terminal differentiation in a variety of solid and haematological cancers in advanced disease patients. In the present study, the LNCaP cell line (prostate cancer) was incubated with SMS or Somadex (an SMS polymer conjugate) for three days, 1 nM per day, and the untreated cells were the negative control. For DNA demethylation, cells were grown in the presence of 2.5 microM 5-aza for 120 h, and re-fed with 5-aza-containing fresh medium at day 3. The total incubation time with 5-aza was 120 h. TSA at 1.0 microM was added into the cultured cells for 24 h. The combined treatment of 5-aza and TSA was performed by incubating the cells with 5-aza for 120 h followed by a 24-h exposure to TSA. Using cDNA obtained from these cell lines, the difference in the expression level of SSTR mRNA transcripts before and after 5-aza and TSA treatments was analyzed by RT-PCR. An increased induction of mRNA expression of the five SSTR subtypes was observed in the LNCaP cells when incubated with SMS/Somadex (dose-dependent). The inhibition of DNA methylation and histone acetylation resulted in the up-regulation of SSTR5 mRNA expression. The results demonstrate a positive feedback loop between SMS and its receptors. This regulation pathway may enhance the anti-tumor activity of somatostatin. To benefit from this effect in a clinical setting, the dose, dose frequency and pan affinity of the SMS derivative are important factors. The epigenetic manipulation with DNA methylation or histone deacetylase inhibitors, combined with SMS, may offer a novel alternative for the treatment of advanced prostate cancer.

Somatostatin effects on the proteome of the LNCaP cell-line.

Some clinical results indicate that somatostatin (sms) might be useful in the treatment of advanced prostate cancer (HRPC). Because of its transient in vivo half-life only more stable derivatives of sms are of interest in this context. Recent studies have shown that natural sms can be conjugated to a carbohydrate (smsdx) with preservation of sms-like effects on the prostatic tumor cell proteome. The present study identifies some of the affected proteins in an effort to elucidate pathways and proteins that might be of importance for the potential usefulness of sms treatment in HRPC. After incubating the LNCaP cell-line with sms14/smsdx, comparative proteomics was used for analysing and identifying affected proteins. Protein expression patterns were analysed with two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. Catalytic mitochondrial and mitochondrial-associated proteins were significantly affected (fold change approximately 2 or higher) and they were in general up-regulated. Apoptosis-related proteins were both up-regulated (VDAC1, VDAC2) and down-regulated (PRDX2, TCTP). The fold change was >2 for PRDX2 and <2 for the others. There was a strong agreement between sms and smsdx on the up- and down-regulation of proteins. Sms/smsdx triggered up-regulation of catalytic mitochondrial proteins and seemed to affect apoptosis-related proteins. This could indicate important pathways on which smsdx might be able to curb the progression of HRPC. The results encourage a pending clinical phase II study.

Prostate cancer cell lines lack amplification: overexpression of HER2.

The potential overexpression of HER2 in prostate cancer cells has attended significant interest during the past few years, both as potential target for HER2 pathway focused therapy and as a mechanism involved in the progression to androgen independence. Conflicting results have been reported concerning HER2 status on clinical material, differences which generally have been attributed to methodological differences. Nevertheless, HER2 has been utilized for targeted therapy of prostate cancer in a number of preclinical studies and is still regarded as an exciting target molecule. In this study, the HER2 status of three widely used prostate cancer cell lines and corresponding xenografts has been analysed. By use of validated and FDA approved analytical staining techniques none of these cell lines or xenografts were shown to overexpress/amplify HER2, as demonstrated by immunohistochemistry and fluorescence in situ hybridization. These findings are important for the interpretation and understanding of the therapeutic effects when developing drugs targeting HER2 in prostate cancer cell lines and also emphasize the importance of using broad and validated analytical techniques.

Proteomic Analysis of a Human Prostate Cancer Cell Line after Incubation with a Novel Somatostatin14 Derivative.

BACKGROUND: Derivatives of somatostatin (sms) are sometimes used in the treatment of hormone-refractory prostatic cancer, in spite of modest results in controlled clinical studies. The optimal use in this context remains to be determined. The human prostatic cancer cell line LNCaP has been used in several previous proteomic analysis studies, which confirmed that sms indeed can affect the protein expression in this cell line. Proteomic analysis is an important tool to increase the understanding of how sms affects the protein expression of the tumor cell. MATERIALS AND METHODS: In this in vitro study, a new sms14 derivative, smsdx, a conjugate between sms14 and dextran, was incubated with an LNCaP cell culture. Sms14 was used as the positive control. Proteomic analysis, using rapid mini two-dimensional electrophoresis, was performed to determine the effects on protein expression. RESULTS: Marked quantitative differences were observed in the protein expression profiles in smsdx-treated LNCaP cells compared to negative control cells (untreated cells). Sets of 63 (yet unidentified) protein spots were differentially expressed. The difference was statistically significant (Mann-Whitney analysis). The 63 dataset was used to accurately discriminate control cells from smsdx-treated cells using hierarchical cluster analysis. Both similarities and differences in protein expression were observed between smsdx- and sms14-treated cells. CONCLUSION: Smsdx is a new sms14 derivative with long in vivo half-life and pan receptor affinity. Sms14-like effects on the protein expression of LNCaP cells seem to be preserved in the construct. The results convey new information about this potentially useful compound. Further studies are now in progress to identify the affected proteins.

Charge-dependent targeting: results in six tumor cell lines.

BACKGROUND: Many previous studies show that cell surface sialylation of malignant cells is enhanced compared to normal tissue. The carboxyl group of the sialic acid yields, a negative surface charge of the tumor cells. This study investigates how tumor cell growth is affected when a cationic polymer is incubated with six different tumor cell lines. MATERIALS AND METHODS: Cationic dextran (CatDex) was prepared by periodate oxidation and subsequent coupling of cationic sidegroups by reductive amination. A fluorimetric cytotoxicity assay (FMCA) was used for the cell survival assay. Six different tumor cell lines (lung, breast, ovarian, prostate, colon, urinary bladder) were seeded into 96-well microtiter plates. CatDex was added at different microM concentrations and incubated for 72 h. Additionally, CatDex was fluorescence-labeled (FITC) and the interaction with the tumor cells was studied using fluorescence microscopy. The presence of sialic acid in the different cell lines was confirmed by using a FITC-labeled sialic acid binding lectin. RESULTS: CatDex showed a concentration-dependent growth inhibitory effect (i.e. the number of cationic side groups/dextran molecule and the molarity used). If the substitution was <20%, the growth inhibitory effect was small and difficult to reproduce. With 20-22% substitution, the growth inhibition varied between 20-95% depending on the molarity and the tumor type. Higher substitution resulted in complete cell death in all the cell lines. The fluorescent images showed intensive cell membrane interaction. CONCLUSION: Incubation with cationic dextran caused cell death in all six tumor cell lines. Our hypothesis is that CatDex binds to the anionic sialic acid residues and causes fatal disturbances in the cell membrane. However the exact mechanism remains to be elucidated. The results may indicate a new method of general interest for intra/local/regiolocal treatment of cancer. Clinical studies to explore this concept are pending.

Development of dextran derivatives with cytotoxic effects in human urinary bladder cancer cell lines.

BACKGROUND: In a previous study we reported on a new approach describing intravesical instillation of charged dextran in patients with superficial bladder carcinoma. The cationic derivative showed a strong tumor-selective accumulation. To develop this approach, the present study investigates the cytotoxic effect of cationic dextran derivatives on two urinary bladder cancer cell lines (J82 and 5637). METHODS: The dextran conjugates were prepared by periodate activation and subsequent coupling by reductive amination. A fluorimetric cytotoxicity assay (FMCA) was used for the cytotoxicity assay. The tumor cells were seeded into 96-well microtiter plates and different cationic dextran derivatives were added and incubated for 72 hours. RESULTS: The results showed that cationic epirubicin-dextran had a clear inhibitory effect on the growth in both cell lines (40-95% growth inhibition). The corresponding values for epirubicin (the reference) was 90-100% inhibition. Interestingly, cationic dextran had, by itself, a growth inhibitory effect. This cytotoxic effect could be strongly enhanced to be almost equal to the reference by changing the cationic sidegroup to aminohexane. Dextran alone showed no effect. CONCLUSION: The finding that cationic dextran by itself can be made cytotoxic, together with its capacity to accumulate in superficial bladder cancer, suggests possibilities for new therapeutic constructs. Cationic dextran with different cationic side-groups and in combination with cytotoxic drugs will be studied further. The cytotoxic mechanism needs to be elucidated.

A novel somatostatin conjugate with a high affinity to all five somatostatin receptor subtypes.

BACKGROUND: Somatostatin receptors (SRS, five subtypes) are expressed in a variety of human tumors, including most tumors of neuroendocrine origin, breast tumors, certain brain tumors, renal cell tumors, lymphomas, and prostate cancer. Somatostatin (SMS) triggers cytostatic and cytotoxic effects and has a general inhibitory effect on secretion mediated through its interaction with SRS. That is the basis for its use in the treatment of SRS-positive tumors. Radiolabeled SMS analogs can also be used for systemic radiotherapy and for diagnostic investigations. METHODS: Sms-14 was conjugated to a periodate-activated dextran70 (mean molecular weight, 70 kD) by reductive amination. The human tumor cell line LCC-18, from a neuroendocrine colonic tumor, was used for stable transfection with each SRS gene separately; transfection was achieved with the expression system TETon (Clontech, Palo Alto, CA). Clones were selected by culturing with G418 and hygromycin B, and positive clones were identified by reverse transcriptase-polymerase chain reaction and binding of iodine-125-labeled SMS-14. The binding affinity for each SRS subtype was then determined for the SMS-dextran conjugate (with SMS-14 used as a positive control). RESULTS: Sms-dextran70 showed high affinity binding to all five receptor subtypes. The IC50 values were between 3 and 80 nM. CONCLUSIONS: This conjugate has a long circulation half-life (i.e., approximately 27 hours after subcutaneous administration in mice) and, with high SRS pan-affinity demonstrated in this study, it has potential in the therapy of SRS-positive tumors. Currently, the clinical significance of SMS-dextran70 is being explored in a clinical Phase I-II study of patients with hormone-refractory prostate cancer. The outcome of this study will be reported when it is available.