Santanu Paul

Curcumin Induces Caspase Mediated Apoptosis in JURKAT Cells by Disrupting the Redox Balance

BACKGROUND Curcumin has has been reported to exert anti-inflammatory, anti-oxidation and anti-angiogenic activity in various types of cancer. It has also been shown to induce apoptosis in leukemia cells. We aimed to unravel the role of the redox pathway in Curcumin mediated apoptosis with a panel of human leukemic cells. MATERIALS AND METHODS In this study in vitro cytotoxicity of Curcumin was measured by MTT assay and apoptotic effects were assessed by annexin V/PI, DAPI staining, cell cycle analysis, measurement of caspase activity and PARP cleavage. Effects of Curcumin on intracellular redox balance were assessed using fluorescent probes like H2DCFDA, JC1 and an ApoGSH Glutathione Detection Kit respectively. RESULTS Curcumin showed differential anti-proliferative and apoptotic effects on different human leukemic cell lines in contrast to minimal effects on normal cells. Curcumin induced apoptosis was associated with the generation of intracellular ROS, loss of mitochondrial membrane potential, intracellular GSH depletion, caspase activation. CONCLUSIONS As Curcumin induces programmed cell death specifically in leukemic cells it holds a great promise as a future therapeutic agent in the treatment of leukemia.

Enhancement of anti-leukemic potential of 2-hydroxyphenyl-azo-2′-naphthol (HPAN) on MOLT-4 cells through conjugation with Cu(II)

A Cu(II) complex of 2-hydroxyphenyl-azo-2′-naphthol (HPAN) having the formula CuII(HPAN)2 was characterized by different techniques. When HPAN and CuII(HPAN)2 were incubated for 24 hours with human T-acute lymphoblastic leukemia (MOLT-4) cells, almost no activity was observed for HPAN while the complex was active. When incubated for 48 hours, HPAN showed cell death of ∼35% at a concentration of 40 μM while CuII(HPAN)2 was only slightly better than when incubated for 24 hours. Therefore, irrespective of incubation time, the anti-proliferative activity due to CuII(HPAN)2 was similar. However, increase in incubation time did show increased activity for HPAN. Anti-leukemic potential was confirmed by microscopic analysis of cell viability by trypan blue stain and MTT assay. The BrdU assay further confirmed proliferative effects of aqueous Cu(II)/HPAN and anti-proliferative effects of Cu(II)(HPAN)2. Propidium iodide staining of Cu(II)(HPAN)2-treated MOLT-4 cells confirmed apoptosis. Since amines formed as a consequence of reduction of the azo bond are reported to be cytotoxic, we performed an enzyme assay to understand the relative reduction of the azo bond in both compounds. Results suggest reduction of the azo bond was slightly higher for HPAN. DNA binding of CuII(HPAN)2 using fluorescence spectroscopy was compared with that of HPAN to determine the propensity of biological activity. The results being similar, binding of the compounds with DNA and the ease of reduction of the azo bond were not able to explain why CuII(HPAN)2 was better in preventing cell proliferation. The high anti-proliferative activity of CuII(HPAN)2 was attributed to increased cellular uptake. We designed experiments to support this hypothesis using independent approaches. In one, Cu(II) was identified in cell lysates using ferrocyanide, while, in another, CuII(HPAN)2 was detected using flow cytometry. We chose Cu(II) as the metal ion for this work because of its recognized involvement in cancer. Being essential for angiogenesis, it is found in increased levels in cancer cells. Interaction of Cu(II)(aq) with MOLT-4 cells confirmed this as a part of this study also. Hence, our objective was to see if molecules like HPAN that bind Cu(II) could lead to its role reversal, i.e. from supporting the growth of cancer cells to be able to destroy them as CuII(HPAN)2.

Identification of a sulfonoquinovosyldiacylglyceride from Azadirachta indica and studies on its cytotoxic activity and DNA binding properties

Chromatographic separation of the methanolic extract of the leaves of Azadirachta indica led to the isolation of a sulfonoglycolipid characterized as a sulfonoquinovosyldiacylglyceride (SQDG), by extensive 2D NMR and mass spectral analysis. SQDG induces apoptosis in a dose dependent manner with IC(50) 8.3 μM against acute lymphoblastic leukemia (ALL) MOLT-4 cell lines. The compound showed significant DNA binding properties as evidenced by the enhancement of melting temperature and perturbation of the characteristic B-form in CD evidence of calf thymus DNA. The DNA binding was also characterized by isothermal calorimetry where a predominantly enthalpy driven binding to CT DNA was revealed.

Acetylation of 1,2,5,8-tetrahydroxy-9,10-anthraquinone Improves Binding to DNA and Shows Enhanced Superoxide Formation that Explains Better Cytotoxicity on JURKAT T Lymphocyte Cells

Background : Hydroxy-9,10-anthraquinones form the core unit of anthracycline anticancer drugs and are close structural analogues to these drugs. Although they show close resemblance to anthracyclines in physicochemical characteristics and electrochemical behavior their biophysical interactions are somewhat weaker than anthracyclines which is a disadvantage. One reason is the formation of anionic species by hydroxy-9,10-anthraquinones. Hence if formation of anionic species is prevented there could be a possibility hydroxy-9,10-anthraquinones would bind DNA better. Procedure : For this 1, 2, 5, 8-tetrahydroxy-9,10-anthraquinone (THAQ) was acetylated to obtain a tetra-acetylated derivative (THAQ-ace) whose interaction with calf thymus DNA was studied using UV-Vis spectroscopy at different pH. Results : Binding constant values for THAQ-ace (~10 5 ) were higher than THAQ at different pH. Increase in binding constant was attributed to anionic species not formed for THAQ-ace at physiological pH. Hence, unlike THAQ, binding constant values for THAQ-ace interacting with calf thymus DNA did not show variation with pH. In fact, it remained more or less constant. Increase in size of the acetylated form (THAQ-ace) compared to THAQ had a negative influence on binding. THAQ-ace showed enhanced superoxide formation. Both DNA binding and superoxide formation were responsible for a significant improvement in anticancer activity for THAQ-ace compared to THAQ on Jurkat T lymphocyte cells . Conclusion : Binding constant values for THAQ-ace binding to DNA were close to that reported for some standard anthracyclines. Hence, suitable modification of the less costly hydroxy-9,10-anthraquinones could provide alternatives to anthracyclines in cancer chemotherapy.

Diterpenoids- Potential Chemopreventive and Chemotherapeutic Agents in Leukemia

Leukemia remains a major cause of death in both developing as well as developed nations worldwide. Development of novel therapeutic approaches is perceived to be the best strategy to combat this disease. Availability of limited effective drugs with least side effects is the major hurdle in treating leukemia. Another major concern remains initiation and engagement of therapeutic strategies that may subvert or overcome drug-resistance. Deployment of plant-based molecules in therapeutic engagements is regarded to be the most apt approach to combat different forms of leukemia. Diterpenoids of natural origin have a broad range of therapeutic activities including anti-leukemic effect. In vitro and in vivo studies establish many diterpenoids from natural sources and their synthetic derivatives to have appreciable cytotoxic and apoptotic efficacies against a series of leukemic cells. This paper attempts to identify and describe diterpenoids with their plant sources and analyze the molecular mechanistic approaches underlying their effective apoptotic activities against leukemic cells studied till date. The present review article summarizes different categories of diterpenoids having various degrees of anti-leukemic activities and discusses the measures of overcoming the bio-availability issues of these natural molecules so that they can serve as extremely viable options in future leukemia therapy.

Role of different aberrant cell signalling pathways prevalent in acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is one of the major forms of leukemia that affects mostly adolescent individuals. The main cause of the development of ALL is not known though several important signal transduction pathways have been reported with functional abnormality in all the cases. Crucial signalling pathways reported in ALL include PI3K/Akt, Notch, Wnt, mTOR, JaK/Stat, etc. Over the past several decades important progress has been made in the management of ALL, however, relapses and post therapy survival ratio has not improved much. This brings the need for understanding the biology and mechanism involved in ALL occurrences and find new molecular targets for better treatment options and risk-adapted therapies to improve the outcome of ALL patients.