Christian Velez

Mitochondrial permeability and toxicity of diethylhexyl and monoethylhexyl phthalates on TK6 human lymphoblasts cells

Phthalates are ubiquitous compounds used in the manufacturing industry. Some are known endocrine disruptors, acting as xenoestrogens, others induce reproductive toxicity and damage to DNA among other effects. Studies on apoptosis induction and mitochondrial damage capacity of phthalates on the immune system are limited. This study aims to determine cell viability inhibition and apoptosis induction of diethylhexyl phthalate (DEHP) and monoethylhexyl phthalate (MEHP) on the human TK6 lymphoblast cell line at concentrations found in the environment. Key hallmark events, such as mitochondrial membrane permeability, generation of reactive oxygen species (ROS) and activation of caspase 3 and 7 were measured. Concentrations that inhibit viability of 50% (IC50) of the cells were determined at 24, 48 and 72 h with doses ranging from 10 to 500 μM. Changes in mitochondrial membrane permeability, ROS generation and activation of caspases 3 and 7, were measured as part of the cell death mechanism. The IC50 at 24 h was approximately 250 μM for both phthalates; at 48 h were 234 and 196 μM for DEHP and MEHP, respectively and at 72 h IC50s were 100 and 80 μM for DEHP and MEHP, respectively. Overall the longer the time of exposure the lower the IC50s for both compounds. Both compounds affected mitochondrial membrane potential, promoted ROS generation and activated caspases 3 and 7. MEHP is more toxic, promotes higher level of ROS production and caspases activation. Our findings suggest that DEHP and MEHP have the capacity to induce apoptosis in cells of the immune system at concentrations found in the environment.

Imidazolium-derived ionic salts induce inhibition of cancerous cell growth through apoptosis

A study of the effects of imidazolium-based ionic liquids on 60 human cancer cell lines representing diverse histologies has identified four compounds which show potency at a nanomolar dose. Their effects on annexin V, DNA fragmentation, and the cell cycle, together with mitochondrial membrane permeabilization tests, provide insights into their mechanism of action. Also, experiments with A431 human epidermoid carcinoma cells suggest the activation of an apoptotic pathway, due to activity of the initiator caspase 8 and effector caspase 3.

Synthesis of novel C5-curcuminoid-fatty acid conjugates and mechanistic investigation of their anticancer activity

The first synthesis of C5-curcumin-fatty acid (C5-Curc-FA) conjugates was successfully performed. Through a two-step synthetic route, 10 analogs were synthesized for a structure-activity relationship (SAR) study. It was found that C5-Curc-FA conjugates containing either decanoic acid or palmitic acid moieties were cytotoxic against colorectal adenocarcinoma cell (CCL-229) at IC50s ranging from 22.5 to 56.1μg/mL, being 5c the most active C5-Curc-FA conjugate. Our results strongly suggests that a decanoic acid moiety at the meta position in C5-Curc-FA conjugates is important for their anticancer activity effect. Possible mechanisms for the anticancer activity of C5-Curc-FA conjugates were also investigated including apoptosis induction, mitochondrial damage and caspases activation. It was shown that 5c inhibited the luminescence activity of NFκB, a key signaling molecule involved in cell apoptosis and cell proliferation, at IC50=18.2μg/mL. In addition, it was demonstrated that 5c displayed significant apoptotic effect at GI50=46.0μg/mL in colorectal adenocarcinoma cell line (ATCC CCL-222), which can be explained by the significant mitochondrial membrane permeabilization and caspases 3 and 7 activation effect of 5c. Finally, it was investigated that C5-Curc-FA conjugates can affect the replication process of cancer cells, since compounds 5c, 5e, and 6c inhibited the relaxing activity of the human DNA topoisomerase I at minimum inhibitory concentrations (MICs) that range from 50 to 250μg/mL. Our results strongly support the hypothesis that the inhibition of both NFκB and DNA topoisomerase I by C5-Curc-FA conjugates is associated with their anticancer activity.

Toxic effects of xylazine on endothelial cells in combination with cocaine and 6-monoacetylmorphine.

The use of xylazine as a drug of abuse has emerged worldwide in the last 7 years, including Puerto Rico. Clinical findings reported that xylazine users present greater physiological deterioration, than heroin users. The aim of this study was to assess the xylazine toxicity on endothelial cells, as this is one of the first tissues impact upon administration. Human umbilical vein endothelial cells in culture were treated with xylazine, cocaine, 6-monoacetylmorphine (heroin metabolite) and its combinations, at concentrations of 0.10-400 μM, for periods of 24, 48 and 72 h. IC50 were calculated and the Annexin V assay implemented to determine the cell death mechanism. Results indicated IC50 values at 24h as follow: xylazine 62 μM, cocaine 210 μM, 6-monoacetylmorphine 300 μM. When these drugs were combined the IC50 value was 57 μM. Annexin V results indicated cell death by an apoptosis mechanism in cells treated with xylazine or in combination. Results demonstrated that xylazine use inhibits the endothelial cell proliferation, at lower concentrations than cocaine and 6-monoacetylmorphine. These findings contribute to the understanding of the toxicity mechanisms induced by xylazine on endothelial cells.

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.

Biological Activity of N-Hydroxyethyl-4-aza-2,3-didehydropodophyllotoxin Derivatives upon Colorectal Adenocarcinoma Cells.

Etoposide is a chemotherapy drug derived from the natural lignin podophyllotoxin. Our novel generated Aza-podophyllotoxin compounds (AZP 8a & AZP 9a) are analogues of podophyllotoxin and were previously screened for anti-cancer activity through the NCI 60 cell line screening panel showing activity on various cell types including colon cancer. This study expands the toxicological screening by studying apoptosis and various hallmark events as part of the mechanism of action of these compounds on colon cancer cells. The COLO 205 cell line was selected and exposed to AZP to determine the IC50 doses at 24 hours treatment. Apoptosis hallmark events such as migration of phosphatidylserine (PS) to the cell membrane, DNA fragmentation, cell cycle effects, mitochondrial membrane permeabilization and caspase activation were included. Experiments were performed in triplicates for all tested compounds including AZP 8a, AZP 9a, camptothecin as positive control and vehicle as negative control. Our results present contrasting apoptotic activity between the experimental compounds. Compound 8a presented migration of PS (annexin V assay), DNA fragmentation and cell cycle arrest at S phase. Compound 9a presented PS migration with fragmented DNA, cell cycle arrest at S phase, mitochondrial membrane permeabilization and activation of caspase 3, 8 and 9. Compound 8a without the oxygen atoms in ring A appears to cause effects similarly to autophagy as induced by etoposide, a cancer drug analogue of our heterocyclic compounds. Compound 9a with the oxygen atoms in expanded ring A presented induction of cell death following activation of a classical apoptosis pathway. Our results suggest that minor structural differences among these AZP can account for the difference in biological response and cancer cell toxicity.

Abstract 1586: Toxicity screening, apoptosis hallmark events and nitro reduction of novel quinolinium salts (BQS) on lymphoma cells

The main purpose of this research was to evaluate the effects of two novel benzazolo[3,2-a]quinolinium salts (ABQ-48 and NBQ48) on lymphoma cells (TOLEDO). The cationic nature of these BQS can facilitate its interaction with cell organelles such as mitochondria and DNA. To determine its potential cytotoxic effects the compounds were screened through the NCI 60 cell line protocol which determines the cell viability inhibitory activity in various cancer histological types. The IC50 dose for these compounds was determined and used to assess their apoptosis induction capacity evaluating apoptosis hallmark events such as DNA Fragmentation, Caspase 3&7 activators and mitochondrial membrane permeabilization. Additionally cell reduction potential of the compounds was measured. Results from the NCI 60 cell line protocol demonstrated the toxicity of BQS in multiple cancer types including breast and colon cancer cells. Preliminary results indicated mitochondrial membrane permeabilization on cells treated with NBQ48 (51.3%) and ABQ48 (57.67%) comparable to the positive controls camptothecin (51.6%) and valinomycin (55.3%). Treated cells also presented DNA fragmentation with NBQ48 and ABQ48 (28%) comparable to the positive control 37%. Preliminary data for caspases 3&7 activation strongly suggest an intrinsic apoptosis pathway. Analysis of the reduction of nitro substituted compound NBQ 48 to the amino substituted ABQ 48 on treated cells was determined by measuring an increment on fluorescence emission, characteristic of an amino specie. In conclusion, the study provides evidence of the induction of key apoptotic events as part of the mechanism of action of these novel BQS. Citation Format: Karoline Rios-Rodriguez, Jessica Soto, Christian Velez, Osvaldo Cox, Juan P. Rivera, Beatriz Zayas. Toxicity screening, apoptosis hallmark events and nitro reduction of novel quinolinium salts (BQS) on lymphoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1586. doi:10.1158/1538-7445.AM2014-1586

Abstract 5157: Preparation of curcumin analog nanoparticles and determination of their anticancer activity in prostate cancer

Prostate cancer is the most frequently diagnosed cancer and the second cause of cancer-related deaths in American men. Bioavailability of therapeutic agents is important for the treatment effectiveness. Previous studies have shown that the development of nanoparticles (NPs) drug delivery vehicles offers an opportunity for targeted drug delivery to tumor cells. Natural compounds such as curcumin have shown decreased bioavailability and stability when used as anticancer agents. Therefore, the purpose of this study is to synthesize the curcumin analog m-nitrochalcone (3bNchalc) encapsulated into poly (lactic-co-glycolic acid) (PLGA) NPs and determine the anticancer activity against prostate cancer cell lines. In our approach, 3bNChalc were encapsulated into PLGA NPs in the presence of PVA using the single emulsion-solvent evaporation method. 3bNChalc loading, encapsulation efficiency, and drug release was determined by spectrophotometric techniques. Physico-chemical properties such as zeta potential, particle size, polydispersity index (PDI), and morphology was measured using a combination of Dynamic Light Scattering (DLS) and Scanning Electron Microscopy (SEM). Cell viability and proliferation of PC3 and 22RV1 prostate cancer cell lines treated with 3bNchalc NPs and control were assessed using the MTS assay. 3bNchalc PLGA NPs were found to have a particle size of 250 nm and smooth spherical shape. In addition, zeta potential and mobility values revealed that 3bNChalc PLGA NPs are negatively charged and they tend to repel each other avoiding the tendency to flocculate. Moreover, 22RV1 cells treated with 3bNchalc PLGA NPs showed decreased in viability and proliferation when compared to control. We demonstrated that 3bNChalc was successfully encapsulated into PLGA NPs. In addition, our results showed that 3bNchalc PLGA NPs decreased the viability of 22RV1 prostate cancer cells when compared to control, suggesting that PLGA improves the delivery of 3bNChalc inside the cell. Results of our study will impact broadly the field by developing more effective and less toxic PLGA NPs based therapies. Citation Format: Maylein C. Juan-Rivera, Maria M. Sanchez-Vazquez, Gamalier Maldonado, Geovanny Ruiz, Noralejandra Vazquez, Christian Velez, Beatriz Zayas, Carlos Cabrera, Magaly Martinez-Ferrer, David Sanabria-Rios. Preparation of curcumin analog nanoparticles and determination of their anticancer activity in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5157. doi:10.1158/1538-7445.AM2017-5157

Abstract 4774: Hypoxic bio-reduction of novel NBQ48 in treated tumor cells and the activity of cytochrome P450 reductase

The main goal of this research was to evaluate the reduction activity of a novel Nitrobenzazolo[3,2-a]quinolinium (NBQ48) compound under hypoxic environment thru the formation of a fluorescent metabolite and further to evaluate the activity of the cytochrome P450 (CYP450) reducatase in the reduction of these compounds. To evaluate the reduction of NBQ48 and the formation of its fluorescent metabolite two (2) tumor cell lines in culture Toledo (lymphoma) and A431 (endothelial) where treated under nitrogen or argon saturated environments to create a hypoxic (low oxygen) environment. In a 15 ml conical tube 4×106 cells in RPMI 1640 media were treated with NBQ 48 (3mM) for a final volume of 5mL and incubated with argon or nitrogen saturated environment at 37oC, 5%. Cells were exposed at different time periods (0 to 48 hours) to evaluate the effect of exposure time in the reduction of the NBQ48 by detecting the reduced fluorescent product, the amino-substituted ABQ48. In addition NBQ48 treated tumor cells were located inside a hypoxic chamber to evaluate the formation of the fluorescent metabolites indicative of a metabolically active cell. To determine if the CYP450 reductase enzyme is activated in the reduction of the NBQ towards the formation of the ABQ metabolite, an experiment containing the NBQs and the CYP reductases was designed. Four (4) samples were prepared: blank media hypoxic, NBQ experimental hypoxic, positive ABQ spiked and negative aerobic control NBQ. Samples with NBQ48 (0.4mM) were incubated for 24 hours. After the incubation period, fluorescence emissions indicating the reductive fluorescent product were measured using a Modulus fluorometer (blue filter). The results on tumor cells treated with NBQ48 under hypoxic conditions demonstrated reduction and the formation of a fluorescent product, amino-substituted ABQ48. The time dependent increase in fluorescence in comparison with non treated cells indicated that the reduction of the NBQ increased with time. Results on the determination of the activity of CYP450 in the reduction of the NBQ48 indicated that only samples containing the CYP450 showed the formation of a fluorescent species presumably, ABQ48. This study provides evidence of the reduction of NBQ48 and the formation of a fluorescent metabolite thru the activations of the CYP450 reductases. The implemented experimental design could also be used to determine activity of hypoxic tissues with clinical applications. Citation Format: Beatriz Zayas, Vivian Lebron, Juan P. Rivera, Christian Velez, Osvaldo Cox. Hypoxic bio-reduction of novel NBQ48 in treated tumor cells and the activity of cytochrome P450 reductase. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4774.

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.