The chemopreventive agent curcumin is a potent radiosensitizer of human cervical tumor cells via increased reactive oxygen species production and overactivation of the mitogen-activated protein kinase pathway.

Cervical cancer is the second most common malignancy among women worldwide and is highly radioresistant, often resulting in local treatment failure. For locally advanced disease, radiation is combined with low-dose chemotherapy; however, this modality often leads to severe toxicity. Curcumin, a polyphenol extracted from rhizomes of the plant Curcuma longa, is a widely studied chemopreventive agent that was shown to have a low toxicity profile in three human clinical trials. Here, we show that pretreatment of two cervical carcinoma cell lines, HeLa and SiHa, with curcumin before ionizing radiation (IR) resulted in significant dose-dependent radiosensitization of these cells. It is noteworthy that curcumin failed to radiosensitize normal human diploid fibroblasts. Although in tumor cells, curcumin did not significantly affect IR-induced activation of AKT and nuclear factor-kappaB, we found that it caused a significant increase in the production of reactive oxygen species, which further led to sustained extracellular signal-regulated kinase (ERK) 1/2 activation. The antioxidant compound N-acetylcysteine blocked the curcumin-induced increased reactive oxygen species (ROS), sustained activation of ERK1/2, and decreased survival after IR in HeLa cells, implicating a ROS-dependent mechanism for curcumin radiosensitivity. Moreover, PD98059 (2'-amino-3'-methoxyflavone)-, PD184352- [2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide], and U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynylthio)butadiene]-specific inhibitors of mitogen-activated protein kinase kinase 1/2 (MEK1/2) blocked curcumin-mediated radiosensitization, demonstrating that the sustained ERK1/2 activation resulting from ROS generation leads to curcumin-mediated radiosensitization. Together, these results suggest a novel mechanism for curcumin-mediated radiosensitization involving increased ROS and ERK1/2 activation and suggest that curcumin application (either systemically or topically) may be an effective radiation modifying modality in the treatment of cervical cancer.

Mechanisms of enhanced neutralization of botulinum neurotoxin by monoclonal antibodies conjugated to antibodies specific for the erythrocyte complement receptor.

Immune complexes formed between monoclonal antibodies (mAbs) and toxins can neutralize toxicity in vivo by multiple mechanisms. Toxin sequestration and clearance by mAbs may be improved by enhancing their ability to bind to red blood cells (RBCs) through immune adherence. This can be achieved by converting the mAbs to heteropolymers (HPs), which are antigen-specific mAbs cross-linked to mAbs targeting the complement receptor (CR1), a protein that is expressed on the surface of RBCs in primates and mediates delivery of complement C3b-containing immune complexes to tissue macrophages. Conversion of mAbs to HPs has been shown to enhance clearance of multivalent antigens from the blood circulation, but the interaction of HPs with monovalent toxins has not been examined. Using botulinum neurotoxin (BoNT) as a model system, we studied the effect of conversion of a pair of BoNT-specific mAbs into HPs on toxin neutralization and handling in vivo. Two HPs given in combination had 166-fold greater potency than un-modified mAbs, neutralizing 5000 LD50 BoNT, when tested in transgenic mice expressing human CR1 on RBC membranes. Improvement required adherence of BoNT to the RBC in vivo and 2 HPs, rather than an HP+mAb pair. The HP pair bound BoNT to RBCs in the circulation for 2h, in comparison to BoNT-neutralizing anti-serum, which induced no detectable RBC binding. HP pairs exhibited enhanced uptake by peritoneal macrophages in vitro, compared to pairs of mAbs or mAb+HP pairs. In a post-exposure therapeutic model, HPs gave complete protection from a lethal BoNT dose up to 3h after toxin exposure. In a pre-exposure prophylaxis model, mice given HP up to 5 days prior to BoNT administration were fully protected from a lethal BoNT dose. These studies elucidate general mechanisms for the neutralization of toxins by HP pairs and demonstrate the potential utility of HPs as BoNT therapeutics.

Enhanced neutralization potency of botulinum neurotoxin antibodies using a red blood cell-targeting fusion protein.

Botulinum neurotoxin (BoNT) potently inhibits cholinergic signaling at the neuromuscular junction. The ideal countermeasures for BoNT exposure are monoclonal antibodies or BoNT antisera, which form BoNT-containing immune complexes that are rapidly cleared from the general circulation. Clearance of opsonized toxins may involve complement receptor-mediated immunoadherence to red blood cells (RBC) in primates or to platelets in rodents. Methods of enhancing immunoadherence of BoNT-specific antibodies may increase their potency in vivo. We designed a novel fusion protein (FP) to link biotinylated molecules to glycophorin A (GPA) on the RBC surface. The FP consists of an scFv specific for murine GPA fused to streptavidin. FP:mAb:BoNT complexes bound specifically to the RBC surface in vitro. In a mouse model of BoNT neutralization, the FP increased the potency of single and double antibody combinations in BoNT neutralization. A combination of two antibodies with the FP gave complete neutralization of 5,000 LD50 BoNT in mice. Neutralization in vivo was dependent on biotinylation of both antibodies and correlated with a reduction of plasma BoNT levels. In a post-exposure model of intoxication, FP:mAb complexes gave complete protection from a lethal BoNT/A1 dose when administered within 2 hours of toxin exposure. In a pre-exposure prophylaxis model, mice were fully protected for 72 hours following administration of the FP:mAb complex. These results demonstrate that RBC-targeted immunoadherence through the FP is a potent enhancer of BoNT neutralization by antibodies in vivo.

Preferential cytotoxicity of bortezomib toward hypoxic tumor cells via overactivation of endoplasmic reticulum stress pathways.

Hypoxia is a dynamic feature of the tumor microenvironment that contributes to drug resistance and cancer progression. We previously showed that components of the unfolded protein response (UPR), elicited by endoplasmic reticulum (ER) stress, are also activated by hypoxia in vitro and in vivo animal and human patient tumors. Here, we report that ER stressors, such as thapsigargin or the clinically used proteasome inhibitor bortezomib, exhibit significantly higher cytotoxicity toward hypoxic compared with normoxic tumor cells, which is accompanied by enhanced activation of UPR effectors in vitro and UPR reporter activity in vivo. Treatment of cells with the translation inhibitor cycloheximide, which relieves ER load, ameliorated this enhanced cytotoxicity, indicating that the increased cytotoxicity is ER stress-dependent. The mode of cell death was cell type-dependent, because DLD1 colorectal carcinoma cells exhibited enhanced apoptosis, whereas HeLa cervical carcinoma cells activated autophagy, blocked apoptosis, and eventually led to necrosis. Pharmacologic or genetic ablation of autophagy increased the levels of apoptosis. These results show that hypoxic tumor cells, which are generally more resistant to genotoxic agents, are hypersensitive to proteasome inhibitors and suggest that combining bortezomib with therapies that target the normoxic fraction of human tumors can lead to more effective tumor control.

Early exposure to methylphenidate increases fear responses in an aversive context in adult rats.

The effects of methylphenidate hydrochloride (MPH) administration during development on fear acquisition and retention in adulthood were examined using classical fear conditioning. Male Sprague-Dawley rats were administered MPH (2 mg/kg) or saline twice daily from postnatal day (PD) 25 to 39, and were trained and tested on PD 81 and 82. During training, shock unconditioned stimulus (US) presentations were explicitly paired with an auditory conditioned stimulus (CS) or occurred unsignaled in the training context. No effect of MPH treatment was found during fear acquisition when shock was signaled or unsignaled during training, but 24 h retention tests in the training context revealed enhanced fear responses in MPH-treated animals that received unsignaled training. These results support recent reports of enhanced anxiety-like behaviors in adult rats caused by early developmental MPH treatment and highlight the need for further research into the long-term effects of developmental exposure to stimulants aimed at pediatric populations.