Toxicity and Apoptosis Related Effects of Benzimidazo [3,2-α] Quinolinium Salts Upon Human Lymphoma Cells.

OBJECTIVES: The present study evaluates novel cationic quinoline derivatives known as benzimidazo[3,2-a]quinolinium salts (BQS) named NBQ-48 and ABQ-48 that have structural similarities to known anti-cancer substances such as ellipticine and berberine. METHODS: Toledo human lymphoma (ATCC CRL2631) cells were treated for 24 to 48 hours. Apoptosis related endpoints such as cell cycle arrest, mitochondrial damage, RNS and ROS generation and the activity of several apoptosis related proteins such as caspases and apoptosis inducing factor (AIF) were studied using fluorescence staining and western blot respectively. RESULTS: Results indicated a higher toxicity from the amino substituted ABQ-48 versus the NBQ-48 (GI50's of 50uM versus 100uM respectively). Both compounds induced cell death through various apoptosis related endpoints including a decrease in mitochondrial membrane potential with an increase in ROS and activation of the effector caspase 3. Interestingly, AIF release was observed on cells treated with the amino substituted ABQ-48 but not on the nitro substituted NBQ-48 samples suggesting a caspase independent mechanism for ABQ-48. CONCLUSIONS: The results obtained presents the toxic effects of two novel benzimidazo[3,2-a]quinolinium salts in human lymphoma tumor cells. The identified mechanism of action includes multiple apoptosis related effects. Furthermore the data presents a clear variation in caspase dependent or independent mechanism for each compound.

Immunomodulatory Response Triggered by the Alkaloids, 3-Amino-7-Benzylbenzimidazo[3,2-a] Quinolinium Chloride (ABQ-48) and 3-Nitro-7-Benzylbenzimidazo [3,2-a] Quinolinium Chloride (NBQ-48).

ABQ-48 (3-amino-7-benzylbenzimidazo[3,2-a]quinolinium chloride) and NBQ-48 (3-nitro-7-benzylbenzimidaw[3,2-a] quinolinium chloride) are un-natural alkaloids containing a planar heteroaromatic systems characterized by quaternized nitrogen fused to benzothiazole nucleus. Both compounds are structurally related to naturally occurring substances such as elliptine (from Ochrosia), and berberine (from Berberis). Previous in vitro studies have shown these agents to control tumor-cell proliferation indicating that both BQS are active but especially ABQ-48 at a 1 OuM dose with over 80% control of the proliferation of multiple cancer cell lines from various etiologies including colon, melanoma, CNS and ovarian cells. Mechanism of action studies have also been conducted however this is the first approach to evaluate immune modulatory activity of these novel BQS. Immune-based therapy is an increasing field in which scientists identify how the immunomodulatory activity of known and newly discovered compounds elicits an immune response that could be used against diseases. In this study, our main objective was to apply an in vitro model to show the immunomodulatory effects of ABQ-48 and NBQ-48 by analyzing the cytokine profile resulting after extracted murine spleen cells were treated with both BQS using a fluorescence-based multiplex ELISA approach. Screened cytokines included: G-CSF, GM-CSF, IL-1a, IL-2, IL-3, IL-5, IL-6, IL-7, IL-10, IL-12p70, IL-13, IL-15, IL-17, IL-21, IL-23, IFN-γ, and TNF-α. Our study results show ABQ 48 and NBQ-48 to stimulate the release of G-CSF, IL-2, IL-6, and, IFN-γ when mouse splenocytes are incubated with serial dilutions of these agents. Our finding opens new possibilities of potentially using ABQ-48 and NBQ-48 as immunomodulatory agents; with intend to activate the immune system such as the production of neutrophils against cancer or reducing chemotherapy side effects.

Comparison of the nucleic acid covalent binding capacity of two nitro-substituted benzazolo[3,2-a]quinolinium salts upon enzymatic reduction.

The DNA binding capacity of two nitro-substituted benzazolo[3,2-a]quinolinium chlorides (NBQs), NBQ-38 and NBQ-95, was evaluated upon their enzymatic reduction with hypoxanthine (HX)/xanthine oxidase (XO) under anaerobic conditions. In the presence of 2'-deoxyguanosine (2'-dG) or calf thymus DNA, covalent-addition products were monitored. Reactions of each NBQ with 2'-dG or DNA differed in the NBQ to HX molar ratio. Control reactions, one without HX/OX and another under aerobic conditions, were also analyzed. Adducts were isolated and characterized by high performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry (ESI-MS). Authentic samples of the reduced forms of these NBQs, identified as ABQ-38 and ABQ-95, were synthesized as standards to monitor bioreduction processes. HPLC analysis showed that the yield of formation of an unknown product (possibly, 2'-dG-NHBQ-38 adduct) from the reaction of NBQ-38 with 2'-dG and DNA was proportional to the HX to NBQ-38 molar ratio. ESI-MS analysis of the DNA hydrolysates showed evidence of an adduct formed upon bioreduction of NBQ-38 by the ions detection at m/z 528.3 and 454.8, consistent with chemical structures of a 2'-dG-NHBQ-38 adduct and a fragment ion. DNA adducts were not observed with NBQ-95, although the corresponding bioreduction product ABQ-95 was detected by ESI-MS. This study provides mechanistic information of these bioreductively-activated pro-drugs with potential therapeutic applications.