5-5--6-6--tetrachloro-1-1--3-3--tetraethylbenzimidazolocarbocyanine and Pancreatic-Neoplasms

Treatment of pancreatic cancer that cannot be surgically resected currently relies on minimally beneficial cytotoxic chemotherapy with gemcitabine. As the fourth leading cause of cancer-related death in the United States with dismal survival statistics, pancreatic cancer demands new and more effective treatment approaches. Resistance to gemcitabine is nearly universal and appears to involve defects in the intrinsic/mitochondrial apoptotic pathway. The bioactive sphingolipid ceramide is a critical mediator of apoptosis initiated by a number of therapeutic modalities. It is noteworthy that insufficient ceramide accumulation has been linked to gemcitabine resistance in multiple cancer types, including pancreatic cancer. Taking advantage of the fact that cancer cells frequently have more negatively charged mitochondria, we investigated a means to circumvent resistance to gemcitabine by targeting delivery of a cationic ceramide (l-t-C6-CCPS [LCL124: ((2S,3S,4E)-2-N-[6'-(1″-pyridinium)-hexanoyl-sphingosine bromide)]) to cancer cell mitochondria. LCL124 was effective in initiating apoptosis by causing mitochondrial depolarization in pancreatic cancer cells but demonstrated significantly less activity against nonmalignant pancreatic ductal epithelial cells. Furthermore, we demonstrate that the mitochondrial membrane potentials of the cancer cells were more negative than nonmalignant cells and that dissipation of this potential abrogated cell killing by LCL124, establishing that the effectiveness of this compound is potential-dependent. LCL124 selectively accumulated in and inhibited the growth of xenografts in vivo, confirming the tumor selectivity and therapeutic potential of cationic ceramides in pancreatic cancer. It is noteworthy that gemcitabine-resistant pancreatic cancer cells became more sensitive to subsequent treatment with LCL124, suggesting that this compound may be a uniquely suited to overcome gemcitabine resistance in pancreatic cancer.

Topics: Animals; Antimetabolites, Antineoplastic; Antineoplastic Agents; Apoptosis; Benzimidazoles; Blotting, Western; Carbocyanines; Cell Death; Cell Line, Tumor; Ceramides; Chromatography, High Pressure Liquid; Coloring Agents; Cytochromes c; Deoxycytidine; Female; Gemcitabine; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Mitochondria; Oxygen Consumption; Pancreatic Neoplasms; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectrum Analysis; Xenograft Model Antitumor Assays

The lichen compound usnic acid is used for its antimicrobial activities in cosmetic products and is also a component of slimming agents. Its effect against cancer cells was first noted over 30 years ago. In this study possible mechanisms of this effect were investigated using two human cell lines, the breast cancer cell line T-47D and the pancreatic cancer cell line Capan-2. Pure (+)-usnic acid from CLADONIA ARBUSCULA and (-)-usnic acid from ALECTORIA OCHROLEUCA were shown to be equally effective inhibitors of DNA synthesis, with IC (50) 4.2 microg/mL and 4.0 microg/mL for (+) and (-)-usnic acid against T-47D, and 5.3 microg/mL and 5.0 microg/mL against Capan-2, respectively. Flow cytometric analysis confirmed the inhibited entry into the S-phase and showed reduction in cell size. Classical apoptosis, as assessed by TUNEL staining, was not observed. Necrosis, measured by LDH release, was seen only in Capan-2 after exposure for 48 hours. Staining with the mitochondrial dye JC-1 demonstrated dose-dependent loss of mitochondrial membrane potential following treatment with usnic acid in both cell lines. In conclusion, usnic acid had a marked inhibitory effect on growth and proliferation of two different human cancer cell lines and led to loss of mitochondrial membrane potential. Cell survival was little affected; late necrosis was seen in one of the cell lines. No difference was noted between the two enantiomers.

Topics: Antineoplastic Agents, Phytogenic; Benzimidazoles; Benzofurans; Breast Neoplasms; Carbocyanines; Cell Cycle; Cell Line, Tumor; Cell Proliferation; DNA; Dose-Response Relationship, Drug; Female; Flow Cytometry; Humans; Inhibitory Concentration 50; Lichens; Membrane Potential, Mitochondrial; Necrosis; Pancreatic Neoplasms; Phytotherapy; Plant Extracts

Resveratrol, a phytoalexin found in the skin of grapes, is believed to have multiple bioactivities including anti-cancer, anti-carcinogenesis and antiinflammatory. The mechanisms by which resveratrol might produce these effects are not well understood. In this study, malignant human pancreatic cancer cells were treated without or with resveratrol in combination with ionizing radiation (IR), and then the mitochondrial function of treated cells was evaluated using several standardized assays. They include the Calcein AM method for mitochondria transition pore; the JC-1 staining method for mitochondria membrane potential; the CM-H2DCFDA method for reactive oxygen species; and the Annexin V/propidium iodide (PI) method for apoptosis/cell death. Our results indicated that (1) pore function was partially intact after resveratrol, but resveratrol probably interfered with the accumulation of intracellular Calcein AM; (2) depolarization of the mitochondria membrane was increased in the resveratrol treated cells, consistent with mitochondrial dysfunction; (3) ROS was slightly increased with resveratrol, a phenomenon that was greatly increased when this agent was combined with IR; and (4) in parallel with the above changes in mitochondrial and drug transport, cells treated with resveratrol showed increased apoptosis as measured by Annexin V/PI staining. In summary, the anti-cancer effect of resveratrol is associated with the damage of mitochondrial function that leads to increased ROS, apoptosis, and possibly intracellular drug accumulation via inhibition of proteins involved in multi-drug resistance (MDR).

Topics: Anticarcinogenic Agents; Apoptosis; Benzimidazoles; Carbocyanines; Cell Death; Cell Line, Tumor; Dose-Response Relationship, Drug; Fluoresceins; Fluorescent Dyes; Humans; Membrane Potentials; Mitochondria; Mitochondrial Membranes; Pancreatic Neoplasms; Reactive Oxygen Species; Resveratrol; Stilbenes

To investigate the effects on human pancreatic cancer PANC-1 and SW1990 cells using a combination of lidamycin (LDM) and gemcitabine.. A 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine the growth inhibition of drugs in PANC-1 and SW1990 cells. The effects on apoptosis were measured by terminal uridine deoxynucleotidyl transferase dUTP nick end labeling assay and flow cytometry combined with fluorescein- isothiocyanate-Annexin V/propidium iodide staining. The activity of caspase-3 was measured with a special assay kit. The mitochondrial membrane potential was determined by confocal microscopy analyses. The level of mRNA encoding K-ras in the cells was determined by RT-PCR analysis. The expression of K-ras, NF-kappaB, and Bcl-2 was detected by Western blotting analysis.. There was a significant reduction in proliferation in the pancreatic cancer cell lines treated with a combination of gemcitabine and LDM. The overall growth inhibition directly correlated with apoptotic cell death. LDM potentiated the gemcitabine-induced cell killing by reducing mitochondrial membrane potential and increasing the caspase-3 activity. Notably, the K-ras mRNA level was significantly reduced with the combination of gemcitabine and LDM. The results for K-ras, NF-kappaB, and Bcl-2 proteins also showed downregulation in the combination group relative to the single-agent treatment and the untreated control.. LDM can potentiate the growth inhibition induced by gemcitabine in human pancreatic cancer cells, and the synergy may be associated with NF-kappaB downregulation.

Topics: Aminoglycosides; Antimetabolites, Antineoplastic; Apoptosis; Benzimidazoles; Carbocyanines; Carcinoma; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Survival; Deoxycytidine; Down-Regulation; Drug Synergism; Enediynes; Fluorescent Dyes; Gemcitabine; Humans; Membrane Potential, Mitochondrial; NF-kappa B; Pancreatic Neoplasms; RNA, Messenger