A unique squalenoylated and nonpegylated doxorubicin nanomedicine with systemic long-circulating properties and anticancer activity.

We identified that the chemical linkage of the anticancer drug doxorubicin onto squalene, a natural lipid precursor of the cholesterol's biosynthesis, led to the formation of squalenoyl doxorubicin (SQ-Dox) nanoassemblies of 130-nm mean diameter, with an original "loop-train" structure. This unique nanomedicine demonstrates: (i) high drug payload, (ii) decreased toxicity of the coupled anticancer compound, (iii) improved therapeutic response, (iv) use of biocompatible transporter material, and (v) ease of preparation, all criteria that are not combined in the currently available nanodrugs. Cell culture viability tests and apoptosis assays showed that SQ-Dox nanoassemblies displayed comparable antiproliferative and cytotoxic effects than the native doxorubicin because of the high activity of apoptotic mediators, such as caspase-3 and poly(ADP-ribose) polymerase. In vivo experiments have shown that the SQ-Dox nanomedicine dramatically improved the anticancer efficacy, compared with free doxorubicin. Particularly, the M109 lung tumors that did not respond to doxorubicin treatment were found inhibited by 90% when treated with SQ-Dox nanoassemblies. SQ-Dox nanoassembly-treated MiaPaCa-2 pancreatic tumor xenografts in mice decreased by 95% compared with the tumors in the saline-treated mice, which was significantly higher than the 29% reduction achieved by native doxorubicin. Concerning toxicity, SQ-Dox nanoassemblies showed a fivefold higher maximum-tolerated dose than the free drug, and moreover, the cardiotoxicity study has evidenced that SQ-Dox nanoassemblies did not cause any myocardial lesions, such as those induced by the free doxorubicin treatment. Taken together, these findings demonstrate that SQ-Dox nanoassemblies make tumor cells more sensitive to doxorubicin and reduce the cardiac toxicity, thus providing a remarkable improvement in the drug's therapeutic index.

Improving the antitumor activity of squalenoyl-paclitaxel conjugate nanoassemblies by manipulating the linker between paclitaxel and squalene.

A series of new lipid prodrugs of paclitaxel, which can be formulated as nanoassemblies, are described. These prodrugs which are designed to overcome the limitations due to the systemic toxicity and low water solubility of paclitaxel consist of a squalene chain bound to the 2'-OH of paclitaxel through a 1,4-cis,cis-dienic linker. This design allows the squalene-conjugates to self-assemble as nanoparticular systems while preserving an efficient release of the free drug, thanks to the dienic spacer. The size, steric hindrance, and functional groups of the spacer have been modulated. All these prodrugs self-assemble into nanosized aggregates in aqueous solution as characterized by dynamic light scattering and transmission electron microscopy and appear stable in water for several days as determined by particle size measurement. In vitro biological assessment shows that these squalenoyl-paclitaxel nanoparticles display notable cytotoxicity on several tumor cell lines including A549 lung cell line, colon cell line HT-29, or KB 3.1 nasopharyngeal epidermoid cell line. The cis,cis-squalenyl-deca-5,8-dienoate prodrug show improved activity over simple 2'-squalenoyl-paclitaxel prodrug highlighting the favourable effect of the dienic linker. The antitumor efficacy of the nanoassemblies constructed with the more active prodrugs has been investigated on human lung (A549) carcinoma xenograft model in mice. The prodrug bearing the cis,cis-deca-5,8-dienoyl linker shows comparable antitumor efficacy to the parent drug, but reveals a much lower subacute toxicity as seen in body weight loss. Thus, nanoparticles with the incorporated squalenoyl paclitaxel prodrug may prove useful for replacement of the toxic Cremophor EL.

Design, optimization and in vitro evaluation of reverse micelle-loaded lipid nanocarriers containing erlotinib hydrochloride.

Erlotinib hydrochloride (ERLO) belongs to the tyrosine kinase inhibitor family and is used for the treatment of pancreatic cancers. In the present study, ERLO was entrapped in lipid nanocarriers by means of reverse micellar incorporation. This study aims to optimize the formulation of ERLO-loaded nanoparticles. Surfactants forming reverse micelles in Labrafac(®) were filled with ERLO under various conditions. Both the initial amount of drug incubated with reverse micelles and the surfactant composing the reverse micelles are crucial parameters for reverse micelle capacity to load ERLO. The optimal loading system for reverse micelles was obtained with a mix of sorbitan trioleate (Span(®) 85) and Labrafac(®) oil at a 1:1 (w/w) ratio. Reverse micelle composition influenced the nanocarrier's hydrodynamic diameter, polydispersity index, and zeta potential. In lipid nanoparticles formulated by using the phase inversion temperature (PIT) method, ERLO entrapment efficiency was around 56%. In vitro, the efficacy of ERLO-loaded nanocarriers on BxPC-3 pancreatic adenocarcinoma cells was comparable to free ERLO, and led to a cell death rate of around 40%.

Squalenoyl nucleoside monophosphate nanoassemblies: new prodrug strategy for the delivery of nucleotide analogues.

4-(N)-1,1',2-trisnor-squalenoyldideoxycytidine monophosphate (SQddC-MP) and 4-(N)-1,1',2-trisnor-squalenoylgemcitabine monophosphate (SQdFdC-MP) were synthesized using phosphoramidite chemistry. These amphiphilic molecules self-assembled to about hundred nanometers size nanoassemblies in aqueous medium. Nanoassemblies of SQddC-MP displayed significant anti-HIV activity whereas SQdFdC-MP nanoassemblies displayed promising anticancer activity on leukemia cells. These results suggested that squalene conjugate of negatively charged nucleotide analogues efficiently penetrated within cells. Thus, we propose a new prodrug strategy for improved delivery of nucleoside analogues to ameliorate their biological efficacy.

Telomerase transcriptional targeting of inducible Bax/TRAIL gene therapy improves gemcitabine treatment of pancreatic cancer.

Currently, gemcitabine is approved as the first-line therapy for patients with locally advanced or metastatic pancreatic cancer. Unfortunately, because of pre-existing or acquired chemoresistance of most of the tumor cells, gemcitabine has failed to significantly improve the outcome for pancreatic carcinoma patients. The present study explored the possibility of sensitizing pancreatic cancer to gemcitabine chemotherapy by combining the chemotherapy with the proapoptotic genes Bax and TNF-related apoptosis-inducing ligand (TRAIL). We designed two tetracycline-inducible recombinant adenoviruses using the human telomerase reverse transcriptase (hTERT) promoter for transcriptional apoptogene targeting. Our data showed that treatment with the adenoviral systems resulted in high-level expression of Bax and TRAIL genes directly related to apoptosis induction, leading to a significant sensitization of resistant pancreatic tumor cells. Furthermore, treatment with Bax and TRAIL adenoviruses plus a suboptimal dose of gemcitabine resulted in significant tumor regression and prolongation of the experimental animal';s life, in contrast to the weak retardation in tumor growth observed when gemcitabine alone was used. Additionally, using an orthotopic tumor model, we showed the usefulness of a non-invasive whole-body optical imaging for real-time evaluation of therapeutic efficacy. Together, these findings suggest that hTERT-targeted proapoptotic gene expression in combination with gemcitabine may be a potential therapeutic strategy for treatment of pancreatic adenocarcinoma.

Efficient electrogene therapy for pancreatic adenocarcinoma treatment using the bacterial purine nucleoside phosphorylase suicide gene with fludarabine.

The aim of this study was to demonstrate the potential of electrogene therapy with the bacterial purine nucleoside phosphorylase gene (ePNP), on pancreatic carcinoma (PC) large tumors. The in vivo electroporation (EP) conditions and efficacy were investigated on both subcutaneous xenografts of human PC cells in immunocompromised mice and orthotopic intrapancreatic grafts of rat PC cells in syngenic rats. After intratumoral injection of naked plasmid DNA, EP was performed using a two-needle array with 25-msec pulses and either a 300 V/cm field strength for subcutaneous or a 500 V/cm field strength for orthotopic PC, parameters providing the best electrotransfer as reflected by the measurements of both luciferase activity and ePNP mRNA. As expected, tumors developed sensitivity to prodrug treatment (6-methylpurine deoxyribose or fludarabine phosphate). We observed both significant inhibition of tumor growth and extended survival of treated mice. In fact, after prodrug treatment, PC growth in the subcutaneous model was delayed by 50-70% for ePNP-expressing tumors. In an orthotopic pancreatic tumor model, the animal survival was significantly prolonged after ePNP electrogene transfer followed by fludarabine treatment, with one animal out of 10 being tumor-free 6 months thereafter. The current study demonstrates for the first time on PC the in vivo feasibility of electrogene transfer and its therapeutic efficiency using the suicide gene/prodrug system, ePNP/fludarabine. These findings suggest that electrogene therapy strategy must be considered for pancreatic cancer treatment, particularly at advanced stages of the disease.

K-ras oncogene silencing strategy reduces tumor growth and enhances gemcitabine chemotherapy efficacy for pancreatic cancer treatment.

Pancreatic adenocarcinoma remains a fatal disease characterized by rapid tumor progression, high metastatic potential and profound chemoresistance. Gemcitabine is the current standard chemotherapy for advanced pancreatic cancer, but it is still far from optimal and novel therapeutic strategies are needed urgently. Mutations in the k-ras gene have been found in more than 90% of pancreatic cancers and are believed to play a key role in this malignancy. Thus, the goal of this study was to investigate the impact of k-ras oncogene silencing on pancreatic tumor growth. Additionally, we examined whether combining k-ras small interfering RNA (siRNA) with gemcitabine has therapeutic potential for pancreatic cancer. The treatment of tumor cell cultures with the corresponding k-ras siRNA resulted in a significant inhibition of k-ras endogenous expression and cell proliferation. In vivo, tumor xenografts were significantly reduced with k-ras siRNA(GAT) delivered by electroporation. Moreover, combined treatment with pSsik-ras(GAT) plus gemcitabine resulted in strong growth inhibition of orthotopic pancreatic tumors. Survival rate was significantly prolonged and the mean tumor volume was dramatically reduced in mice receiving the combined treatment compared with single agents. Collectively, these findings show that targeting mutant k-ras through specific siRNA might be effective for k-ras oncogene silencing and tumor growth inhibition. The improvement of gemcitabine-based chemotherapy suggests that this strategy might be used therapeutically against human pancreatic cancer to potentiate the effects of conventional therapy.

Valproic acid induces non-apoptotic cell death mechanisms in multiple myeloma cell lines.

Multiple myeloma (MM) is an incurable hematological disorder characterized by dysregulated proliferation of terminally differentiated plasma cells. Aberrant histone acetylation has been observed in the development of numerous malignancies. Histone deacetylase inhibitors such as valproic acid (VPA) are promising drugs for cancer therapy since they have been reported to have antiproliferative effects and to induce differentiation in carcinoma and leukemic cells. Considering the advantage of being already in clinical use for epilepsy treatment, valproic acid might be a promising therapeutic candidate drug in the management of multiple myeloma. In this study, we show that the short fatty acid VPA has a time and dose-dependent cytotoxic effect on the MM cell lines OPM2, RPMI and U266. The influence of VPA on cell cycle and apoptosis have been evaluated by flow cytometry. Our results show that the three cell lines are blocked in G0/G1 phase. The observed sensitivity to VPA can be partially explained by late apoptosis. Since caspase 3 is activated in all tested cell lines after VPA treatment, a caspase-dependent pathway seems to be involved but not activated by the classic apoptotic pathways. We have also studied another mechanism of cell death, the senescence-like phenotype, but did not find any evidence for its implication. Thus, treatment with VPA may imply other alternative cell death mechanisms.

Transcriptional tumor-selective promoter targeting of E. coli purine nucleoside phosphorylase for pancreatic cancer suicide gene therapy.

BACKGROUND: Pancreatic cancer remains a rapidly fatal disease. Suicide gene therapy has been shown to be an effective tool for pancreatic tumor cell destruction, but a cell-specific gene delivery is required to limit host toxicity. The objective of this study was both to design recombinant vectors in which the suicide gene E. coli purine nucleoside phosphorylase (ePNP) is under the control of either CEA or MUC1 promoter sequences and to investigate on experimental pancreatic carcinomas the selective killing effects of the conditional ePNP/prodrug (MePdR) system. METHODS: Transcriptional activities of CEA and MUC1 promoter sequences were analyzed using luciferase reporter gene constructions. Thereafter, recombinant vectors expressing ePNP under control of the most promising pCEA and pMUC1 sequences were designed and used to establish stable tumor cell transfectants from two human pancreatic cell lines, respectively tumor-marker positive (BxPc3) or negative (Panc-1), then applied for in vitro and in vivo experiments. RESULTS: Transient experiments indicated that CEA and MUC1 promoter sequences confer specificity while preserving high transcriptional activities. The MePdR treatment induced a high in vitro cytotoxicity on the sole CEA- and MUC1-producing cell lines (i.e. BxPc3-CEA and -MUC1/ePNP). In the same way, prodrug treatment induced a significant tumor regression on the sole tumor-marker-positive BxPc3 xenografts, whilst the Panc1-CEA and -MUC1/ePNP tumors were not affected. CONCLUSIONS: These data confirm and extend the antitumor efficacy of the ePNP/MePdR killing system and demonstrate the feasibility of the transcriptional targeting strategy under tumor marker promoter control and thereby a preferential killing of CEA- and MUC1-producing pancreatic tumor cells. Thus, efficient in vivo gene delivery and transcriptional targeting constitute the major future clinical challenge for a selective pancreatic cancer suicide gene strategy.

Suicide gene/prodrug therapy for pancreatic adenocarcinoma by E. coli purine nucleoside phosphorylase and 6-methylpurine 2'-deoxyriboside.

OBJECTIVE: Recent advances in diagnostics, staging, and therapy for pancreatic cancer have not resulted in significant improvements in long-term survival, and development of new approaches is particularly urgent. The use of prodrug-activating genes is a possible approach for cancer gene therapy. The aim of this study was to evaluate the efficacy of Escherichia coli purine nucleoside phosphorylase (ePNP) on pancreatic tumors. ePNP activates the prodrug 6-methylpurine deoxyribose (MePdR) into methyl purine (MeP), which is highly toxic to dividing and nondividing cells. METHODS: A recombinant pCAG-ePNP vector was constructed and used to establish pancreatic cancer cells expressing constitutively ePNP (ePNP+). The ePNP/MePdR system effects were tested in vitro on HA-RPC (rat) and BxPC3 (human) pancreatic cancer cell lines and then in vivo on tumors established in nude mice with BxPC3 ePNP+ cells. RESULTS: MePdR treatment of ePNP+ tumor cells induced cytotoxic and antiproliferative effects in a concentration-dependent manner with a 100% cell death since 5 x 10 mol/L. Bystander effect was strong in vitro as more than 50% of tumor cells were killed by MePdR with only 1%-2% of ePNP+ cells. In vivo, tumor growth was completely abolished with a prodrug treatment initiated 2 days after tumor cell inoculation, and mice remained tumor free. In addition, even if MePdR treatment was applied to large tumors, tumors significantly regressed. CONCLUSION: These preliminary results support the therapeutic potential of the MePdR/ePNP system, which induces a highly cytotoxic effect with a potent bystander effect on pancreatic tumors.

Combined suicide gene therapy for pancreatic peritoneal carcinomatosis using BGTC liposomes.

Peritoneal dissemination is a common end-stage complication of pancreatic cancer for which novel therapeutic modalities are actively investigated, as there is no current effective therapy. Thus, we evaluated, in a mouse model of pancreatic peritoneal carcinomatosis, the therapeutic potential of a novel nonviral gene therapy approach consisting of bis-guanidinium-tren-cholesterol (BGTC)-mediated lipofection of a combined suicide gene system. Human BxPC-3 pancreatic cells secreting the carcinoembryonic antigen (CEA) tumor marker were injected into the peritoneal cavity of nude mice. After 8 days, intraperitoneal (i.p.) lipofection was performed using BGTC/DOPE cationic liposomes complexed with plasmids encoding the two prodrug-activating enzymes Herpes Simplex Virus thymidine kinase and Escherichia coli cytosine deaminase, the latter being expressed from a bicistronic cassette also encoding E. coli uracil phosphoribosyltransferase. Administration of the lipoplexes was followed by treatment with the corresponding prodrugs ganciclovir and 5-fluorocytosine. The results presented herein demonstrate that BGTC/DOPE liposomes can efficiently mediate gene transfection into peritoneal tumor nodules. Indeed, HSV-TK mRNA was detected in tumor nodule tissues by semiquantitative reverse transcription-polymerase chain reaction analysis. In addition, green fluorescent protein (GFP) fluorescence and X-gal staining were observed in the peritoneal tumor foci following lipofection of the corresponding EGFP and LacZ reporter genes. These expression analyses also showed that transgene expression lasted for about 2 weeks and was preferential for the tumor nodules, this tumor preference being in good agreement with the absence of obvious treatment-related toxicity. Most importantly, mice receiving the full treatment scheme (BGTC liposomes, suicide genes and prodrugs) had significantly lower serum CEA levels than those of the various control groups, a finding indicating that peritoneal carcinomatosis progression was strongly reduced in these mice. In conclusion, our results demonstrate the therapeutic efficiency of BGTC-mediated i.p. lipofection of a combined suicide gene system in a mouse peritoneal carcinomatosis model and suggest that BGTC-based prodrug-activating gene therapy approaches may constitute a potential treatment modality for patients with peritoneal carcinomatosis and minimal residual disease.

Feasibility, sensitivity, and reliability of laser-induced fluorescence imaging of green fluorescent protein-expressing tumors in vivo.

Whole-body imaging of green fluorescent protein (GFP) can be used to test the efficiency of gene carriers for in vivo transduction. The aim of the current study was to determine the sensitivity and the accuracy of a GFP imaging procedure by in vivo investigation of GFP-expressing tumor cells. An improved method of whole-body GFP imaging made use of a laser excitation source and band-pass filters matched specifically to GFP and constitutive tissue fluorescence emission bands. Processing of the primary GFP fluorescence images acquired by the CCD camera subtracted background tissue autofluorescence. Our approach achieved 100% sensitivity and specificity for in vivo detection of 10%-transfected BxPc3 pancreatic tumor after subcutaneous grafting or orthotopical implantation in the pancreas of nude mice. It also detected less transfected tumors (i.e., 1 to 5%) but with a loss in sensitivity (50% of cases). The system was employed over a 5-week period to monitor the persistence of GFP expression in 10%-transfected BxPc3 tumors orthotopically implanted in the pancreas of two nude mice, allowing the direct visualization of tumor progression and spread. In facilitating the temporal-spatial follow-up of GFP expression in vivo, the optimized laser-induced fluorescence imaging device can support preclinical investigations of vectors for therapeutic gene transduction through regular, harmless, real-time monitoring of theirin vivo transductional efficacy and persistence.