The variable chemotherapeutic response of Malabaricone-A in leukemic and solid tumor cell lines depends on the degree of redox imbalance

Purpose: The ‘two-faced’ character of reactive oxygen species (ROS) plays an important role in cancer biology by acting as secondary messengers in intracellular signaling cascades, enhancing cell proliferation and survival, thereby sustaining the oncogenic phenotype. Conversely, enhanced generation of ROS can trigger an oxidative assault leading to a redox imbalance translating into an apoptotic cell death. Intrinsically, cancer cells have higher basal levels of ROS which if supplemented by additional oxidative insult by pro-oxidants can be cytotoxic, an example being Malabaricone-A (MAL-A). MAL-A is a plant derived diarylnonanoid, purified from fruit rind of the plant Myristica malabarica whose anti-cancer activity has been demonstrated in leukemic cell lines, the modality of cell death being apoptosis. This study aimed to compare the degree of effectiveness of MAL-A in leukemic vs. solid tumor cell lines.Methods: The cytotoxicity of MAL-A was evaluated by the MTS-PMS cell viability assay in leukemic cell lines (MOLT3, K562 and HL-60) and compared with solid tumor cell lines (MCF7, A549 and HepG2); further studies then proceeded with MOLT3 vs. MCF7 and A549. The contribution of redox imbalance in MAL-A induced cytotoxicity was confirmed by pre-incubating cells with an antioxidant, N-acetyl-l-cysteine (NAC) or a thiol depletor, buthionine sulfoximine (BSO). MAL-A induced redox imbalance was quantitated by flow cytometry, by measuring the generation of ROS and levels of non protein thiols using dichlorofluorescein diacetate (CM-H2DCFDA) and 5-chloromethylfluorescein diacetate (CMFDA) respectively. The activities of glutathione peroxidase (GPx), superoxide dismutase, catalase (CAT), NAD(P)H dehydrogenase (quinone 1) NQO1 and glutathione-S-transferase GST were measured spectrophotometrically. The mitochondrial involvement of MAL-A induced cell death was measured by evaluation of cardiolipin peroxidation using 10-N-nonyl acridine orange (NAO), transition pore activity with calcein-AM, while the mitochondrial transmembrane electrochemical gradient (∆ψm) was measured by JC-1, fluorescence being acquired in a flow cytometer. The apoptotic mode of cell death was evaluated by double staining with annexin V-FITC and propidium iodide (PI), cell cycle analysis by flow cytometry and caspase-3 activity spectrophotometrically. The expression of Nrf2 and HO-1 was examined by western blotting. Results: MAL-A demonstrated a higher degree of cytotoxicity in three leukemic cell lines whose IC50 ranged from 12.70 ± 0.10 to 18.10 ± 0.95 µg/ml, whereas in three solid tumor cell lines, the IC50 ranged from 28.10 ± 0.58 to 55.26 ± 5.90 µg/ml. This higher degree of cytotoxicity in MOLT3, a leukemic cell line was due to a higher induction of redox imbalance, evident by both an increased generation of ROS and concomitant depletion of thiols. This was confirmed by pre-incubation with NAC and BSO, wherein NAC decreased MAL-A induced cytotoxicity by 2.04 fold while BSO enhanced MAL-A cytotoxicity and decreased the IC50 by 5.60 fold. However, in solid tumor cell lines (MCF7 and A549), NAC minimally decreased MAL-A induced cytotoxicity, and BSO increased the IC50 by 1.96 and 2.39 fold respectively. Furthermore, the generation of ROS by MAL-A increased maximally in MOLT3 as the fluorescence increased from 44.28 ± 7.85 to 273.99 ± 32.78, and to a lesser degree in solid tumor cell lines, MCF7 (44.28 ± 14.89 to 207.97 ± 70.64) and A549 (37.87 ± 3.24 to 147.12 ± 38.53). In all three cell lines there was a concomitant depletion of thiols as in MOLT3, the GMFC decreased from 340.65 ± 60.39 to 62.67 ± 11.32, in MCF7 (277.82 ± 50.32 to 100.39 ± 31.93) and in A549 (274.05 ± 59.13 to 83.15 ± 21.43). In MOLT3 as compared to MCF7 and A549, decrease in the activities of GPx, CAT, NQO1 and GST was substantially greater. In all cell lines, the MAL-A induced redox imbalance translated into triggering of initial mitochondrial apoptotic events. Here again, MAL-A induced a higher degree of cardiolipin peroxidation in MOLT3 (67.01%) than MCF7 and A549 (29.15% and 44.30%), as also down regulated the mitochondrial transition pore activity from baseline to a higher extent, GMFC being 48.05 ± 2.37 to 10.70 ± 3.97 (MOLT3), 43.55 ± 3.36 to 15.36 ± 0.60 (MCF7) and 39.58 ± 0.4 to 12.65 ± 1.56 (A549). Perturbation of mitochondrial membrane potential evident by a decrease in the ratio of red/green (J-aggregates/monomers) was 134 fold (14.73/0.11) in MOLT3, 45 fold in MCF7 (20.72/0.46) and 34 fold in A549 (22.01/0.64). The extent of apoptosis using a similar concentration of MAL-A was maximal in MOLT3, wherein a 105 fold increase in annexin V binding was evident (0.83 ± 0.51 to 87.08 ± 9.85%) whereas it increased by 43.11 fold in MCF7 (0.69 ± 0.30 to 29.75 ± 11.79%) and 47.52 fold in A549 (0.61 ± 0.31 to 28.99 ± 17.21%). MAL-A induced apoptosis was also associated with a higher degree of caspase-3 activity in MOLT3 vs. MCF7 or A549 which translated into halting of cell cycle progression, evident by an increment in the sub-G0/G1 population [19.26 fold in MOLT3 (0.95 ± 0.45 vs. 18.30 ± 1.90%), 11.01 fold in MCF7 (0.97 ± 0.37 vs. 10.68 ± 0.69%) and 8.58 fold in A549 (1.06 ± 0.45 vs. 9.10 ± 1.05%)]. MAL-A effectively inhibited Nrf2 and HO-1, more prominently in MOLT3. Furthermore, the decreased expression of Nrf2 in MOLT3 correlated with the decreased activities of NQO1 and GST, suggesting that targeting of the Nrf2 anti-oxidant pathway could be considered. Conclusion: Taken together, MAL-A a pro-oxidant compound is likely to be more effective in leukemias, meriting further pharmacological consideration

Impact of MAPK and PI3K/AKT signaling pathways on Malabaricone-A induced cytotoxicity in U937, a histiocytic lymphoma cell line

Intrinsically cancer cells have higher basal levels of reactive oxygen species (ROS), which when augmented by pro-oxidants such as Malabaricone-A (MAL-A) triggers apoptotic cell death, secondary to ‘turning on’ of the apoptosis related cell signaling pathways. The effects of MAL-A upon key inflammation related signaling molecules were evaluated by western blotting in U937, a histiocytic lymphoma derived cell line. The impact of inhibitors of the pro-apoptotic MAPK and anti-apoptotic PI3K/AKT signaling pathways upon MAL-A induced cytotoxicity and generation of ROS was evaluated by a cell viability assay and flow cytometry respectively in two hematopoietic cell lines, U937 and MOLT3. MAL-A enhanced phosphorylation of the components of the pro-apoptotic pathway, namely ASK1, p38 and JNK. Alongside, MAL-A decreased the phosphorylation of AKT and mTOR. The cytotoxicity of MAL-A was attenuated by inhibitors of p38 and JNK, whereas its cytotoxic potential was enhanced in the presence of a PI3K/AKT inhibitor. Similarly, MAL-A mediated generation of ROS was decreased by inhibitors of p38MAPK and JNK, whereas the PI3K/AKT inhibitor potentiated its generation of ROS. Taken together, MAL-A mediated its cytotoxicity by enhanced generation of ROS via modulation of the apoptosis related cellular signaling pathways and tilting the balance towards a pro-apoptotic scenario. This was achieved via an up-regulation of MAPK (p38 and JNK) along with down-regulation of the PI3K/AKT/mTOR pathway indicating that manipulation of these pathways by compounds such as MAL-A are promising therapeutic targets, worthy of future pharmacological consideration

Allylpyrocatechol attenuates collagen-induced arthritis via attenuation of oxidative stress secondary to modulation of the MAPK, JAK/STAT, and Nrf2/HO-1 pathways

Rheumatoid arthritis (RA), an inflammatory autoimmune disorder is characterized by synovial hyperplasia and bony destruction. The pathogenesis of RA includes redox dysregulation, concomitant with increased levels of pro-inflammatory mediators. As the ability of Allylpyrocatechol (APC), a phytoconstituent of Piper betle leaves to alleviate oxidative stress has been demonstrated in patients with RA, its anti-arthritic activity was evaluated in an animal model of arthritis, along with establishment of the underlying mechanism(s) of action. The animal model was established by immunizing rats with bovine collagen type II (CII) followed by lipopolysaccharide, along with a booster dose of CII on day 15. Rats were treated with APC or Methotrexate (MTX) from days 11 to 27, wherein paw edema, radiography, histopathology and markers of inflammation were evaluated. The pro/anti-inflammatory signaling pathways were studied in a RAW264.7 macrophage cell line. APC prevented the progression of arthritis as evident from the reduction in paw edema, attenuation of damage to bones and cartilage as evident by radiography as also histopathology. Additionally, there was reduction in the levels of pro-inflammatory cytokines (TNF-α and IL-6) and restoration of the redox balance. Importantly, MTX ameliorated the features of arthritis, but not the associated oxidative stress. In RAW264.7, APC inhibited generation of nitric oxide and pro-inflammatory cytokines (TNF-α, IL-6 and IL-12p40), modulated the phosphorylation of pro-inflammatory (ERK1/2, SAPK/JNK, and JAK/STAT) and cytoprotective (Nrf2, HO-1) signaling pathways. Taken together, APC controlled the development of arthritis possibly via modulation of signaling pathways and deserves further consideration in the therapy for RA

Allylpyrocatechol Attenuates Collagen-Induced Arthritis via Attenuation of Oxidative Stress Secondary to Modulation of the MAPK, JAK/STAT, and Nrf2/HO-1 Pathways

ABSTRACT Rheumatoid arthritis (RA), an inflammatory autoimmune disorder, is characterized by synovial hyperplasia and bony destruction. The pathogenesis of RA includes redox dysregulation, concomitant with increased levels of proinflammatory mediators. As the ability of allylpyrocatechol (APC), a phytoconstituent of Piper betle leaves, to alleviate oxidative stress has been demonstrated in patients with RA, its antiarthritic activity was evaluated in an animal model of arthritis, and the underlying mechanism(s) of action clarified. The animal model was established by immunizing rats with bovine collagen type II (CII) followed by lipopolysaccharide, along with a booster dose of CII on day 15. Rats were treated with APC or methotrexate (MTX) from days 11 to 27, when paw edema, radiography, histopathology, and markers of inflammation were evaluated. The pro/antiinflammatory signaling pathways were studied in a RAW264.7 macrophage cell line. Allylpyrocatechol (APC) prevented the progression of arthritis as was evident from the reduction in paw edema, and attenuation of damage to bones and cartilage shown by radiography and histopathology. Additionally, there was reduction in the levels of proinflammatory cytokines [tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6)] and restoration of the redox balance. Importantly, MTX ameliorated the features of arthritis but not the associated oxidative stress. In RAW264.7, APC inhibited generation of nitric oxide and proinflammatory cytokines (TNF-a, IL-6, and IL-12p40), and modulated the phosphorylation of proinflammatory (extracellular signal-regulated kinase 1/2, stress-activated protein kinase/c-Jun N-terminal protein kinase, and Janus kinase/signal transducers and activators of transcription) and cytoprotective (nuclear factor erythroid 2-related factor 2, heme oxygenase-1) signaling pathways. Taken together, APC controlled the development of arthritis, possibly via modulation of signaling pathways, and deserves further consideration as a therapy for RA

Activation of Anthracene Endoperoxides in Leishmania and Impairment of Mitochondrial Functions

Leishmaniasis is a vector-borne disease caused by protozoal Leishmania. Because of resistance development against current drugs, new antileishmanial compounds are urgently needed. Endoperoxides (EPs) are successfully used in malaria therapy, and experimental evidence of their potential against leishmaniasis exists. Anthracene endoperoxides (AcEPs) have so far been only technically used and not explored for their leishmanicidal potential. This study verified the in vitro efficiency and mechanism of AcEPs against both Leishmania promastigotes and axenic amastigotes (L. tarentolae and L. donovani) as well as their toxicity in J774 macrophages. Additionally, the kinetics and radical products of AcEPs’ reaction with iron, the formation of radicals by AcEPs in Leishmania, as well as the resulting impairment of parasite mitochondrial functions were studied. Using electron paramagnetic resonance combined with spin trapping, photometry, and fluorescence-based oximetry, AcEPs were demonstrated to (i) show antileishmanial activity in vitro at IC50 values in a low micromolar range, (ii) exhibit host cell toxicity in J774 macrophages, (iii) react rapidly with iron (II) resulting in the formation of oxygen- and carbon-centered radicals, (iv) produce carbon-centered radicals which could secondarily trigger superoxide radical formation in Leishmania, and (v) impair mitochondrial functions in Leishmania during parasite killing. Overall, the data of different AcEPs demonstrate that their structures besides the peroxo bridge strongly influence their activity and mechanism of their antileishmanial action

Evaluation of serological markers to monitor the disease status of Indian post kala-azar dermal leishmaniasis

Post kala-azar dermal leishmaniasis (PKDL), a dermal sequel of visceral leishmaniasis presents with macular or polymorphic lesions. As immunological variations between these two forms have not been delineated, we evaluated levels of antileishmanial total Ig, IgG and its subclasses, IgM, IgE, IgG avidity, cytokines IL-10, IL-4, IL-13 and expression of CD19. The levels of Ig and IgG in polymorphic PKDL were higher than macular PKDL, while significant curtailment in levels of Ig, IgM and IgG following treatment was evident only in polymorphic PKDL. With regard to IgG subclasses, IgG1 and IgG3 were significantly raised in polymorphic PKDL, whereas in macular PKDL only IgG1 was elevated; treatment decreased levels of IgG1, IgG2 and IgG3 only in polymorphic PKDL; IgE levels were raised in both groups but no marked alterations occurred following treatment. The avidity of IgG was higher in polymorphic PKDL and correlated with duration of disease. IL-10 was higher in polymorphic PKDL and decreased significantly after treatment, whereas in macular PKDL IL-4 predominated. Taken together, in PKDL the humoral immune response was greater in the polymorphic variant than the macular form suggesting that serological markers may have a role in monitoring polymorphic PKDL