This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The overall goal of this project is to further understand the mechanism of toxicity of Benzazolo[3,2-a]quinolinium Salts (BQs) in tumor cells and compare its cytotoxicity to the ones observed on normal T lymphocytes as surrogate of effect for other normal tissues. Within the cytotoxic effects, mitochondrial damage, apoptosis, ROS release and endoplasmic reticulum stress of BQs are included. Known anticancer drugs including etoposide and taxol often present the capability of multiple mechanisms of actions. Preliminary studies with A431 cells have indicated the capacity of BQs to inhibit cellular growth, interact with mitochondria and induce apoptosis similarly to Cisplatin. Additional studies however are necessary to further understand the cell death pathway of cells exposed to BQs. The compounds under study, are nitro and amino containing heterocyclic compounds possessing a positive charge that could facilitate their interaction with cell organelles specially mitochondria. Two of the compounds under study are amino derivatives with fluorescence activity facilitating the observation of their cellular site of action. The cationic nature of these BQs has been implicated in preferential intramitochondrial retention, which allows selective killing of cancer cells. In addition, substituent effects in these compounds may confer differential toxicity. Preliminary results suggested that the BQs can induce apoptosis on A431 cells through the intrinsic pathway involving mitochondrial membrane permeability and activating the effectors caspases 3 &7. Results also suggest the inactivity of caspase 8 in the apoptotic pathway. In this study we will also analyze the toxicity of BQs on normal T lymphocytes as surrogate of effect for other normal cells. Comparison with normal cells is crucial in the process of identifying appropriate chemo therapeutic agents. Understanding how normal lymphocytes react to BQs'exposure as potential anti tumor agent will provide information regarding the selectivity of BQs toward tumor cells preferentially and the identification of early molecular events that could serve as predictive markers of BQs therapeutic outcomes. Hypothesis 1: Structural similarities with known anti tumor compounds suggest that the BQs understudy (NBQ-38, ABQ-38, NBQ-95, ABQ-95 and BQ-108) can induce apoptosis on A431 through an activation pathway that can include the following mechanisms: Mitochondrial damage;Cytochrome c release;Caspase activation;endoplasmic reticulum stress and or generation of reactive oxygen species.
Aim 1 : Determine the apoptosis induction and mitochondrial interaction of BQs in tumor cell lines. The mitochondrial Cytochrome c release on tumor cells will also be determined as confirmation of mitochondrial damage associated with apoptosis.
Aim 2 : Determine the activation of Caspases 3, 7 and 8 as a way of determining the extrinsic or intrinsic apoptotic pathway.
Aim 3 : Determine the generation of ROS as part of the mechanism of cell death on tumor cells treated with BQs.
Aim 4 : Determine the capacity of BQs to induce stress to the reticulum endoplasmic of tumor cells. Modifications have been made to the study aims. A new aim has been included at this stage:"""""""" To determine the DNA fragmentation capacity of BQs in tumor cells"""""""". This new aim was not included in the original proposal since we did not have the appropriate instrumentation at the time of submission. It is however relevant in the characterization of the mechanism of apoptosis induced by these novel substances since DNA fragmentation can be expexted as part of the apoptosis proces. The purpose of this experiment was to evaluate the capacity of BQs to induce DNA fragmentation in A431 tumor cells at the determined IC50. Hypothesis 2: BQs can induce higher toxicity to cancer cell lines than to normal lymphocytes cells through the induction of apoptosis and mitochondria! damage.
Aim 5. Determine the cytotoxic dose response of BQs on human normal TK-6 lymphocytes.
Aim 6. Determine the apoptosis induction and mitochondrial interaction of BQs in human TK6 lymphoblast cells in culture.
Aim 7. Determine through statistical analysis the significant difference in dose response effect between tumor and normal cell exposed to BQs.
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