Enhanced antitumor efficacy of gemcitabine-loaded temperature-sensitive liposome by hyperthermia in tumor-bearing mice.

INTRODUCTION: Gemcitabine-loaded TSL (Gem-TSL) was used in combination with hyperthermia (HT) to treat the colon adenocarcinoma-bearing BALB/c mice for improved anticancer effect following intravenous administration. METHODS: A new temperature-sensitive liposome (TSL), composed of DPPC:DMPC:DSPC (4:1:1 molar ratio) releasing the encapsulated gemcitabine (Gem) at 41 °C, was developed and evaluated for enhanced antitumor efficacy both in vitro and in vivo. RESULTS: Drug release from the TSL was sharply increased at 41 °C and in vitro cytotoxicity of Gem-TSL in colon adenocarcinoma cells (CT-26) was 10 times higher than the free drug (IC50 = 0.3 µM versus 3 µM). Apoptosis seemed to be the main mechanism of cell death as the treatment of the cells with Gem-TSL increased the caspse-3/7 activity by 1.5-fold and also caused the fragmentation of chromatin DNA. Gem-TSL suppressed the tumor growth in CT-26-bearing BALB/c mice more stronger than the free gemcitabine after intravenous administration. Moreover, this in vivo antitumor efficacy of Gem-TSL was further increased when HT was added. DISCUSSION: This study suggests that this new TSL-Gem formulation could serve as a new chemotherapy modality together with HT.

In vivo antitumor effect of cromolyn in PEGylated liposomes for pancreatic cancer.

A PEGylated liposomal formulation of cromolyn, composed of dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-mPEG2000), has been developed with the purpose of improving the antitumor activity of cromolyn for human pancreatic adenocarcinoma. In stability study, the amount of proteins adsorbed onto the PEGylated liposomes encapsulating cromolyn was 4.5-fold lower than the non-PEGylated liposome. In vitro study showed that the cromolyn in PEGylated liposome exhibited better anti-proliferative effect in BxPC-3 cells than in Panc-1 cells, which indicates higher level of endogenous S100P protein in BxPC-3 cells than in Panc-1 cells as a target protein for this drug. Moreover, the combination of cromolyn with gemcitabine in PEGylated liposomes demonstrated the strongest cytotoxicity to BxPC-3 pancreatic cancer cells in vitro and the highest anti-tumor activity against the BxPC-3 tumor bearing nude mice in vivo. Thus, this PEGylated liposomal formulation of cromolyn is expected to provide a novel approach to the treatment of pancreatic cancer in the future.

Enhancement of radiotherapeutic effectiveness by temperature-sensitive liposomal 1-methylxanthine.

Most of methylxanthine derivatives including caffeine have been known to radiosensitize cancer cells, but the obstacles such as toxicity, request of high dose and poor solubility hinder their preclinical evaluations and clinical applications. In this study, we evaluated the efficacy of 1-methylxanthine (1-MTX), a caffeine metabolite as a radiosensitizer and the in vivo effectiveness of the temperature-sensitive liposomal 1-methylxanthine (tsl-MTX) in combination with ionizing radiation and regional hyperthermia. In human colorectal and lung cancer cells, treatment of 1-MTX sensitized cells to ionizing radiation. To evaluate the in vivo capability of 1-MTX to radiosensitize tumors, we developed temperature-sensitive liposomal 1-MTX using DPPC:DMPC:DSPC (4:1:1 molar ratio) with intention of overcoming lethal toxicity of 1-MTX and controlling drug-release. The particle size of the liposomes was approximately 200 nm in diameter. The release of 1-MTX from the liposomes was responding to increase of temperature. In xenograft tumor-bearing mice, the tsl-MTX administered using the i.p. route showed delay of tumor growth. Importantly, tsl-MTX in combination with radiation and regional hyperthermia exhibited marked delay of tumor growth, suggesting that 1-MTX effectively enhanced radiation-induced suppression of tumor growth. In conclusion, tsl-MTX has highly efficacious anticancer competence in vivo, enhancing radiotherapeutic effectiveness, and feasibility for further clinical applications.

Preparation of pH-sensitive, long-circulating and EGFR-targeted immunoliposomes.

A long-circulating formulation of pH-sensitive liposomes (PSLs) with antibodies against epidermal growth factor receptor (EGFR) attached was designed, expecting an increase in binding and delivery of liposomes to the target cells including non-small cell lung cancer (NSCLC) cells. Physicochemical properties of the PSLs were measured by SEM and DLS. Leakage of a self-quenching fluorescent probe, calcein, from the liposome was studied for the evaluation of pH-sensitivity. Encapsulation efficiency of gemcitabine (an anti-cancer drug) in PSLs was about 67%. Average size of liposomes was 88 nm in diameter. The PSL of DOPE/CHEMS (6:4 molar ratio) formulation showed a dramatic pH-sensitivity at/around pH 5.5, whereas non-PSL of DPPC/Chol or PC/CHEMS formulation did not. Anti-proliferation effect of gemcitabine-encapsulating PSLs & Ab-PSLs in A549 cells was 2-fold higher than the free drug, which was further elucidated by the apoptosis of the cells by gemcitabine (approximately 10% apoptosis for PSL or Ab-PSL formulation vs. approximately 1% for free drug or non-PSL formulation) using FACS analysis. These data demonstrate delivery of gemcitabine to tumor cells can be improved by long-circulating PSLs or Ab-PSLs formulations in vitro.

Three novel VHL germline mutations in Korean patients with von Hippel-Lindau disease and pheochromocytomas.

Von Hippel-Lindau (VHL) disease is an autosomal dominant inherited tumor syndrome associated with germline mutations in the VHL gene. VHL disease, as well as several other cancer syndromes, has been associated with an increased risk of pheochromocytomas, which are catecholamine-secreting tumors of the adrenal gland. VHL disease genotype-phenotype correlations have been well established based on the type of mutations in the VHL gene. However, although many groups have reported VHL germline mutations in different countries, no previous report has described VHL gene mutations in VHL disease and/or pheochromocytoma patients in Korea. In this study, we used direct sequencing to investigate VHL germline mutations in Korean patients with VHL disease or pheochromocytomas (11 VHL patients and 3 additional members from 7 families, 2 patients from 1 family with familial pheochromocytoma, and 2 cases of isolated pheochromocytoma). We found a total of 7 VHL germline mutations (6 missense and 1 frameshift), 3 of which were novel (323_324delGC, 355T>C and 361G>A). No VHL germline mutation was found in the 2 patients with isolated pheochromocytomas and paragangliomas. This study provides informative data for VHL germline mutations and VHL-related phenotypes in Korea.

A TP53-truncating germline mutation (E287X) in a family with characteristics of both hereditary diffuse gastric cancer and Li-Fraumeni syndrome.

Mutations in CDH1, which encodes E-cadherin, have been associated with hereditary diffuse gastric cancer (HDGC) in Western populations but have not been shown to play a major role in Asians. Recently, a patient with familial gastric cancer (FGC) was shown to harbor a germline mutation in the TP53 gene, which encodes p53 and has been previously associated with Li-Fraumeni Syndrome (LFS). To determine whether mutations in TP53 are associated with FGC in Asians, we screened the entire coding region of TP53 in probands from 23 Korean FGC families. We identified a nonsense (E287X) TP53 germline mutation in a family whose history is compatible with both HDGC and LFS. Two members of this family (SNU-G2) were afflicted with brain tumors, seven with gastric cancers, two with sarcomas, and one with both gastric cancer and a sarcoma. The E287X TP53 mutation segregated with the cancer phenotype in the family members from whom DNA samples were available. To our knowledge, this is the first report of a large family with both HDGC and LFS. Our results suggest that TP53 mutational screening in FGC families should be interpreted with caution because additional TP53 mutation-carrying HDGC families may also show LFS-related phenotypes.