Synthesis of Gold Nanoparticle: Peptide-Drug Conjugates for Targeted Drug Delivery.

Peptide-drug conjugates (PDCs) are being developed for the targeted delivery of drugs to cancer cells. Several approaches are being followed to enhance their stability in biological solutions. Here we describe an effective method to easily couple PDCs to polyethylene-coated gold nanoparticles. We also outline analytical methods to validate the coupling and assays to measure the stability and cytotoxic efficacy of the conjugates.

Long-term survival without graft-versus-host-disease following infusion of allogeneic myeloma-specific Vß T cell families.

BACKGROUND: Despite chemo-induction therapy and autologous stem cell transplantation (ASCT), the vast majority of patients with Multiple Myeloma (MM) relapse within 7 years and the disease remains incurable. Adoptive Allogeneic T-cell therapy (ATCT) might be curative for MM, however current ATCT protocols often lead to graft versus host disease (GvHD). Transplanting only tumor reactive donor T cells that mediate a graft-versus-myeloma (GvM) but not GvHD may overcome this problem. METHODS: We used an MHC-matched/miHA-disparate B10.D2 → Balb/c bone marrow transplantation (BMT) murine model and MOPC315.BM MM cells to develop an ATCT protocol consisting of total body irradiation, autologous-BMT and infusion of selective, myeloma-reactive lymphocytes of T cell receptor (TCR) Vß 2, 3 and 8.3 families (MM-auto BMT ATCT). RESULTS: Pre-stimulation ex vivo of allogeneic T cells by exposure to MOPC315.BM MM cells in the presence of IL-2, anti-CD3 and anti-CD28 resulted in expansion of the myeloma-reactive T cell TCRVß 2, 3 and 8.3 subfamilies. Their isolation and infusion into MM-bearing mice resulted in a vigorous GvM response without induction GvHD and long-term survival. Repeated infusion of naïve myeloma-reactive T cell TCRVß 2, 3 and 8.3 subfamilies was also effective. CONCLUSIONS: These data demonstrate that a transplantation protocol involving only selective tumor-reactive donor T cell families is an effective immunotherapy and results in long-term survival in a mouse model of human MM. The results highlight the need to develop similar ATCT strategies for MM patients that result in enhanced survival without symptoms of GvHD.

New somatostatin-drug conjugates for effective targeting pancreatic cancer.

Pancreatic cancer poorly responds to available drugs, and finding novel approaches to target this cancer type is of high significance. Here, based on a common property of pancreatic cancer cells to express somatostatin receptors (SSTR), we designed drug conjugates with novel somatostatin-derived cyclic peptides (SSTp) with broad selectivity towards SSTR types to facilitate drug targeting of the pancreatic cancer cells specifically. Uptake of our newly designed SSTps was facilitated by SSTRs expressed in the pancreatic cancers, including SSTR2, SSTR3, SSTR4 and SSTR5. Three major drugs were conjugated to our best SSTps that served as delivery vehicles, including Camptothecin (CPT), Combretastatin-4A (COMB) and Azatoxin (AZA). All designed drug conjugates demonstrated penetration to pancreatic cancer cell lines, and significant toxicity towards them. Furthermore, the drug conjugates specifically accumulated in tumors in the animal xenograft model, though some accumulation was also seen in kidney. Overall these findings lay the basis for development of novel drug series that could target the fatal pancreatic cancer.

Discovery of peptide drug carrier candidates for targeted multi-drug delivery into prostate cancer cells.

Metastatic castration-resistant prostate cancer (mCRPC) remains essentially incurable. Targeted Drug Delivery (TDD) systems may overcome the limitations of current mCRPC therapies. We describe the use of strict criteria to isolate novel prostate cancer cell targeting peptides that specifically deliver drugs into target cells. Phage from a libraries displaying 7mer peptides were exposed to PC-3 cells and only internalized phage were recovered. The ability of these phage to internalize into other prostate cancer cells (LNCaP, DU-145) was validated. The displayed peptides of selected phage clones were synthesized and their specificity for target cells was validated in vitro and in vivo. One peptide (P12) which specifically targeted PC-3 tumors in vivo was incorporated into mono-drug (Chlorambucil, Combretastatin or Camptothecin) and dual-drug (Chlorambucil/Combretastatin or Chlorambucil/Camptothecin) PDCs and the cytotoxic efficacy of these conjugates for target cells was tested. Conjugation of P12 into dual-drug PDCs allowed discovery of new drug combinations with synergistic effects. The use of strict selection criteria can lead to discovery of novel peptides for use as drug carriers for TDD. PDCs represent an effective alternative to current modes of free drug chemotherapy for prostate cancer.

Discovery of potent molecular chimera (CM358) to treat human metastatic melanoma.

The resistance of cancer cells to chemotherapeutic agents, whether through intrinsic mechanisms or developed resistance, motivates the search for new chemotherapeutic strategies. In the present report, we demonstrate a facile synthetic strategy towards the discovery of new anti-cancer substances. This strategy is based on simple covalent coupling between known anti-cancer drugs, which results in novel 'chimeric' small molecules. One of these novel compounds, CM358, is the product of an amide bond formation between the known Topoisomerase II (Topo II) inhibitor amonafide (AM) and the known DNA mustard alkylator chlorambucil (CLB). It demonstrates significant enhanced cytotoxicity over an equimolar mixture of AM and CLB in various cancer cell lines and in a xenograft model of human metastatic melanoma. Topo II inhibition as well as in silico docking studies suggest that CM358 is a stronger Topo II binder than AM. This may be attributed, at least partially, to the placement of the CLB moiety in a favorable orientation with respect to DNA cross-linking with nearby guanines. In a human metastatic melanoma (WM 266-4) xenograft model, this compound was profoundly superior to a mixture of AM and CLB in reduction of tumor growth, maintenance of body weight and extension of overall survival.

MS-KIF18A, a kinesin, is associated with estrogen receptor.

The study of MS-KIF18A kinesin protein is focused on its cellular distribution and association with a cargo protein. Indirect immunofluorescence (IF) analyzed the intracellular distribution of endogenous MS-KIF18A and the transfected enhanced green fluorescence protein (eGFP)-MS-KIF18A in osteogenic cells. In both cases, the proteins were localized at the plasma membrane, cytosol, and nucleus. Bioinformatics analysis suggested interactions between MS-KIF18A and estrogen receptor (ERalpha) which were further elucidated by immunoprecipitation (IP). We identified interaction between endogenous MS-KIF18A with 66 and 46 kDa isoforms of ERalpha in MBA-15 cells. Moreover, MS-KIF18A and 66 kDa ERalpha complex has been demonstrated between ectopically expressed proteins in COS-7 cells. We have shown that anti-MS-KIF18A antibody immunoprecipitated the ERalpha and pERK in cells challenged with 17beta-estrogen (17beta-E2). The hormone activation induced mitogen-activated protein kinases (MAPK) pathway and increased p-ERK. The activation was interfered when cells were pre-treated with either ICI-182,780 or MAPK inhibitor PD98059 prior the challenge with 17beta-E2 that resulted in a decrease in association between MS-KIF18A and p-ERK1/2. The obtained results suggest a role for the proteins in a non-genomic response of MBA-15 cells challenged with 17beta-E2. This study presents a novel interaction between MS-KIF18A and ER that may have important physiological and pharmacological implications for estrogen action in various cells.

MS-KIF18A, new kinesin; structure and cellular expression.

The present study describes the cloning and molecular analysis of a new gene, MS-KIF18A, a member of the kinesin family. MS-KIF18A was cloned from a marrow stromal cells expression library. Transcripts derived from this gene were also detected in testis and trachea, although they differed from the stroma mesenchymal cell transcript in the open reading frame (ORF) as well as in the untranslated regions (UTRs). The existence of various transcripts suggests alternative regulation of gene expression and defines tissue specific function of the new kinesin. The cDNA from the marrow stroma, MS-KIF18A, encodes a predicted protein of 898 amino acids with a molecular weight of 100 kDa. Kinesins are motor proteins that consist of a motor domain with microtubule-binding and ATPase sites, a coiled coil region and a cargo-binding domain. Examination of a three-dimensional model of the MS-KIF18A motor domain suggested that this protein associates with microtubules, which was confirmed by immunofluorescence (IF) experiments in stromal cells.

Elevated expression of the CC chemokine regulated on activation, normal T cell expressed and secreted (RANTES) in advanced breast carcinoma.

Breast carcinoma is the most common malignant disease among women and the second most lethal one. In search for a better understanding of the role of cellular mediators in the progression of this disease, we investigated the potential involvement of the CC chemokine Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES) in breast carcinoma progression. To this end, RANTES expression was determined in breast tumor cell lines and in sections of breast carcinomas, followed by analysis of the incidence and intensity of its expression in different stages of the disease. Our study reveals that high and physiologically relevant levels of RANTES are constitutively produced by T47D and MCF-7 breast tumor cell lines. Analysis of RANTES expression in sections of breast carcinomas demonstrates a high incidence of RANTES expression in epithelial tumor cells; the chemokine was expressed in 74% of the sections. RANTES expression was rarely detected in normal duct epithelial cells or in epithelial cells that constitute benign breast lumps, which were located in proximity to tumor cells. High incidence and intensity of RANTES expression were detected in sections of most of the patients with stage II and stage III of the disease (expression was detected in 83 and 83.3%, respectively), whereas RANTES was expressed at a lower incidence and intensity in sections of patients with stage I of breast carcinoma (55% of the cases). Most importantly, the expression of RANTES was minimally detected in sections of patients diagnosed with benign breast disorders and of women that underwent reduction mammoplasty (15.4% of the cases). These results indicate that the expression of RANTES is directly correlated with a more advanced stage of disease, suggesting that RANTES may be involved in breast cancer progression. Moreover, it is possible that in patients diagnosed with benign breast disorders, RANTES expression may be indicative of an ongoing, but as yet undetectable, malignant process.