PAN-811 Blocks Chemotherapy Drug-Induced In Vitro Neurotoxicity, While Not Affecting Suppression of Cancer Cell Growth.

Chemotherapy often results in cognitive impairment, and no neuroprotective drug is now available. This study aimed to understand underlying neurotoxicological mechanisms of anticancer drugs and to evaluate neuroprotective effects of PAN-811. Primary neurons in different concentrations of antioxidants (AOs) were insulted for 3 days with methotrexate (MTX), 5-fluorouracil (5-FU), or cisplatin (CDDP) in the absence or presence of PAN-811·Cl·H2O. The effect of PAN-811 on the anticancer activity of tested drugs was also examined using mouse and human cancer cells (BNLT3 and H460) to assess any negative interference. Cell membrane integrity, survival, and death and intramitochondrial reactive oxygen species (ROS) were measured. All tested anticancer drugs elicited neurotoxicity only under low levels of AO and elicited a ROS increase. These results suggested that ROS mediates neurotoxicity of tested anticancer drugs. PAN-811 dose-dependently suppressed increased ROS and blocked the neurotoxicity when neurons were insulted with a tested anticancer drug. PAN-811 did not interfere with anticancer activity of anticancer drugs against BNLT3 cells. PAN-811 did not inhibit MTX-induced death of H460 cells but, interestingly, demonstrated a synergistic effect with 5-FU or CDDP in reducing cancer cell viability. Thus, PAN-811 can be a potent drug candidate for chemotherapy-induced cognitive impairment.

Immunoblotting and immunodetection.

Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps, starting with solubilization of the protein samples, usually by means of SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining with Ponceau S. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. After nonspecific binding sites are blocked, the membrane is probed with the primary antibody and washed. The antibody-antigen complexes are tagged with horseradish peroxidase or alkaline phosphatase coupled to a secondary anti-IgG antibody, and detected using appropriate chromogenic or luminescent substrates. Finally, membranes may be stripped and reprobed.

Nicotine vaccines.

Smoking is a global healthcare problem. Current smoking cessation rates using behavioral counseling and pharmacotherapeutic interventions have had modest success, with ∼1:5 smokers remaining abstinent long-term. Nicotine vaccines are a new class of immunotherapeutics under development. It is believed that anti-nicotine antibodies arising from vaccination capture nicotine and prevent or reduce its entry into the brain, as the antibody-bound nicotine is too large to cross the blood-brain barrier. This in turn decreases the pleasurable effects of smoking, reducing or eliminating positive reinforcement, thereby making it easier for a smoker to quit smoking. Four vaccine candidates have advanced into clinical testing with mixed success. Proof-of-concept has been established in that individuals with higher levels of anti-nicotine antibodies were observed to have higher smoking cessation and abstinence rates. Recently, the most advanced candidate vaccine, NicVAX, failed to meet the primary endpoint in two large phase III studies, although the correlation of higher abstinence rates in subjects with higher immunity to nicotine was observed. Although the field has had setbacks, the magnitude of the tobacco epidemic and the positive pre-clinical research and observed clinical trends indicate continued research is warranted. Several avenues are being actively pursued: a) improving vaccine potency by introducing novel carriers and/or adjuvants to stimulate higher immune response b) targeting subjects who have a robust response (e.g. personalized medicine) c) combining vaccines with pharmacotherapy for maintenance of abstinence/relapse prevention.

Immunoblotting and immunodetection.

Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps, starting with solubilization of the protein samples, usually by means of SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining with Ponceau S. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. After nonspecific binding sites are blocked, the membrane is probed with the primary antibody and washed. The antibody-antigen complexes are tagged with horseradish peroxidase or alkaline phosphatase coupled to a secondary anti-IgG antibody, and detected using appropriate chromogenic or luminescent substrates. Finally, membranes may be stripped and reprobed.

Immunoblotting and immunodetection.

Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps starting with solubilization of the protein samples, usually with SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining or Ponceau S staining. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. After nonspecific binding sites are blocked, the membrane is probed with the primary antibody and washed. The antibody-antigen complexes are tagged with horseradish peroxidase or alkaline phosphatase coupled to a secondary anti-IgG antibody, and detected using appropriate chromogenic or luminescent substrates. Finally, membranes may be stripped and reprobed.

Immunoblotting and immunodetection.

Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides numerous protocols for all steps starting with solubilization of the protein samples, usually with SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are then electrophoretically transferred to a membrane, a process that can be monitored by reversible staining or Ponceau S staining. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. Any remaining binding sites are blocked by immersing the membrane in a blocking solution. After probing with the primary antibody, the membrane is washed and the antibody-antigen complexes are identified with horseradish peroxidase (HRPO) or alkaline phosphatase enzymes coupled to the secondary anti-IgG antibody (e.g., goat anti-rabbit IgG) and appropriate chromogenic or luminescent substrates. Finally, membranes may be stripped and reprobed.

Immunoblotting and immunodetection.

Immunoblotting (often referred to as western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit presents procedures for electrophoretically transferring antigens from a denaturing polyacrylamide gel in a tank or a semidry transfer apparatus to a nitrocellulose, PVDF, or nylon membrane. The process can be monitored by reversible staining or by Ponceau S staining, both of which are described here. A protocol for blotting previously stained gels is also described. The transferred proteins are bound to the surface of the membrane, providing access for reaction with immunodetection reagents. All remaining binding sites are blocked by immersing the membrane in a solution containing either a protein or detergent blocking agent. After probing with the primary antibody, the membrane is washed and the antibody-antigen complexes are identified with horseradish peroxidase (HRPO) or alkaline phosphatase enzymes coupled to the secondary anti-IgG antibody (e.g., goat anti-rabbit IgG). The enzymes are attached directly or via an avidin-biotin bridge to the secondary antibody, and protocols are provided for both methods. Chromogenic or luminescent substrates are then used as described to visualize the activity. Finally, a method for stripping and reprobing membranes is presented.