Selective cytotoxicity of 3-amino-l-tyrosine correlates with peroxidase activity

Summary In the presence of 3-amino-l-tyrosine (3-AT), abundant brown pigment forms in human HL-60 cells, but not in a variety of other cell lines, which are reported to be lower in mean myeloperoxidase (MPO) content than HL-60. Cells were assessed for peroxidase activity with an ABTS-based colorimetric assay and compared to values obtained with known amounts of human myeloperoxidase. HL-60 cells were estimated to contain the equivalent of 37.1 ng myeloperoxidase/10⁶ cells versus 26.1 and 5.0 ng/10⁶ cells for human K562 and murine RAW 264.7 cell lines, respectively. HL-60 cells exhibited a nearly 60% inhibition of proliferation and >70% reduction in cell viability after 4 d of culture in the presence of 100 μg 3-AT per ml. Higher concentrations of 3-AT (up to 400 μg/ml) for 4 d reduced HL-60 proliferation by 80% and decreased viability to 1–3%. Comparable levels of cytotoxicity were achieved in KG-1 cells after 7 d with 200 or 400 μg 3-AT per ml. K562 cells exhibited a 40% reduction in cell number after 7 d with 400 μg 3-AT per ml, but concentrations less than 400 μg/ml did not significantly affect K562 proliferation. K562 viability remained unchanged with doses of 3-AT up to 400 μg/ml. RAW 264.7 cells exhibited unchanged viability and proliferation in the presence of 3-AT at concentrations up to 400 μg 3-AT per ml. K562, KG-1, and RAW 264.7 cells exhibited no evidence of brown pigment formation in the presence of 3-AT and medium containing 10% fetal bovine serum. However, RAW 264.7 cells that were converted to protein-free medium and exposed to 3-AT exhibited intense brown pigment in some cell nuclei. A high percentage of HL-60 cells treated with 3-AT exhibited membrane blebbing, pyknosis, and nuclear fragmentation, which was not observed among other 3-AT-treated cell lines. A mechanism involving toxic intermediates of peroxidase-mediated “aminomelanin” formation is hypothesized.     

Dephosphorylation, Proteolysis, and Reduced Activity of Poly(A) Polymerase Associated with U937 Cell Apoptosis

The apoptotic trend of the widely used cell lines HL-60, U937, HeLa, Molt-3, and K562 has been found to be accompanied and reversibly related with Poly(A) polymerase (PAP; EC 2.7.7.19) activity levels. Moreover, variations in the pattern of multiple enzyme forms are revealed, being most prominent in apoptosis-prone cell lines, HL-60 and U937. Furthermore, in heat-shocked or nutrient-deprived apoptotic U937 Percoll-fractionated subpopulations, PAP lower mobility phosphorylated forms of 106 and 100 kDa as well as enzyme activity were progressively reduced along with the appearance of higher than 80 kDa mobility species. The kinetics of these alterations (dephosphorylation, proteolysis, and activity) coincided with the appearance of DNA fragmentation. In fact, PAP dephosphorylation appears to precede the appearance of DNA fragmentation. In addition, inhibition of PAP dephosphorylation, proteolysis, and decrease in its activity were tightly coupled with the concomitant prevention of apoptosis. This novel finding yields information on a possible involvement of PAP in cell commitment and execution to apoptosis.     

The ability to cleave 28S ribosomal RNA during apoptosis is a cell-type dependent trait unrelated to DNA fragmentation

Discrete cleavages within 28S rRNA divergent domains have previously been found to coincide with DNA fragmentation during apoptosis. Here we show that rRNA and DNA cleavages can occur independently in apoptotic cells, i.e. that the previously observed correlation is likely to be coincidental. In HL-60 cells, apoptosis with massive DNA fragmentation could be induced without any signs of rRNA cleavage. The opposite situation; rRNA cleavage without concomitant internucleosomal DNA fragmentation, was found in okadaic acid-treated Molt-4 cells. Other leukemia cell lines underwent apoptosis either without (K562 and Molt-3) or with (U937) both forms of polynucleotide cleavage. In K562 cells transfected with a temperature-sensitive p53 mutant, internucleosomal DNA fragmentation but not 28S rRNA cleavage was inducible by wild-type p53 expression. The absence of apoptotic rRNA cleavage in some cell types suggests that this phenomenon is tightly regulated and unrelated to DNA fragmentation or a presumed scheme for general macromolecular degradation in apoptotic cells.     

Synthesis of Bcl-2 in response to anthracycline treatment may contribute to an apoptosis-resistant phenotype in leukemic cell lines.

BACKGROUND: Some forms of chemoresistance in leukemia may start from failure of tumour cells to successfully undergo apoptosis and Bcl-2 may play a role in this defect. Therefore, we evaluated the Bcl-2 content and synthesis in relation with the apoptotic potential in leukemic cell lines after anthracycline treatment. METHODS: U937, HL60, and K562 cells and their drug resistant (DR) variants were treated with varying concentrations of Idarubicin (IDA). Apoptosis was evaluated by fluorescence microscopy after acridine orange staining. Bcl-2 and Bax content were evaluated either by flow cytometry after indirect immunolabelling or by Western blot. RESULTS: High Bcl-2 contents were not related to a poor ability to undergo apoptosis in U937, HL60, K562 and their DR variants. IDA induced a concentration-dependent increase in Bcl-2 content in all cell lines as long as they do not perform apoptosis. Enhanced Bcl-2 expression was inhibited by cycloheximide, actinomycin D, or antisense oligonucleotide directed against bcl-2 mRNA. Bcl-2 expression was also increased in the resistant U937 variant after serum deprivation or C2-ceramide treatment. The synthesis of Bcl-2 led to an increased Bcl-2/Bax ratio solely in the cells with an apoptosis-resistance phenotype. CONCLUSIONS: These data suggest that exposure to IDA induces Bcl-2 expression in leukemic cell lines, and that this mechanism could contribute to apoptosis resistance and participate in the acquisition of chemoresistance. They also confirm that the evolution of the Bcl-2/Bax ratio reflects apoptotic ability better than the steady state level of Bcl-2 expression.     

Anti-apoptotic role of focal adhesion kinase (FAK). Induction of inhibitor-of-apoptosis proteins and apoptosis suppression by the overexpression of FAK in a human leukemic cell line, HL-60

Focal adhesion kinase (FAK) has an anti-apoptotic role in anchorage-dependent cells via an unknown mechanism. To elucidate the role of FAK in anti-apoptosis, we have established several FAK cDNA-transfected HL-60 cell lines and examined whether FAK-transfected cells have resistance to apoptotic stimuli. FAK-transfected HL-60 (HL-60/FAK) cells were highly resistant to apoptosis induced with hydrogen peroxide (1 mm) and etoposide (50 microg/ml) compared with the parental HL-60 cells or the vector-transfected cells, when determined using viability assay, DNA fragmentation, and flow cytometry analysis. Because no proteolytic cleavage of pro-caspase 3 to mature caspase 3 fragment was observed in HL-60/FAK cells, FAK was presumed to inhibit an upstream signal pathway leading to the activation of caspase 3. HL-60/FAK activated the phosphatidylinositide 3'-OH-kinase-Akt survival pathway and exhibited significant activation of NF-kappaB with marked induction of inhibitor-of-apoptosis proteins (IAPs: cIAP-1, cIAP-2, XIAP), regardless of the hydrogen peroxide-treated or untreated conditions, whereas no significant IAPs were detected in the parental or vector-transfected HL-60 cells. Apoptotic agents induced higher NF-kappaB activation in HL-60/FAK cells than in HL-60/Vect cells, and it appeared that sustained NF-kappaB activation is critical to the anti-apoptotic states in HL-60/FAK cells. Mutagenesis of FAK cDNA revealed that Y397 and Y925, which are involved in the tyrosine-phosphorylation sites, were prerequisite for the anti-apoptotic activity as well as induction of IAPs, and that K454, which is involved in the kinase activity, was also required for the full anti-apoptotic activity of FAK. Taken together, we have demonstrated definitively that FAK-transfected HL-60 cells, otherwise sensitive to apoptosis, become resistant to the apoptotic stimuli. We conclude that FAK activates the phosphatidylinositide 3'-OH-kinase-Akt survival pathway with the concomitant activation of NF-kB and induction of IAPs, which ultimately inhibit apoptosis by inhibiting caspase-3 cascade.     

Lineage-related sensitivity to apoptosis in human tumor cells undergoing hyperthermia

In this study the role of hyperthermia as an apoptotic trigger was analyzed in four human tumor cell lines: HL60, U937, DOHH₂, and K562. These cell lines were chosen because of their well known and different expression of bcl-2 and bcr-abl genes, the expression of which is known to be an antiapoptotic condition. HL60 and U937 cells were strongly susceptible to heat exposure, while DOHH₂ cells were weakly sensitive and K562 cells were resistant, thus suggesting a possible gene involvement in this type of programmed cell death. The mechanisms underlying this apoptosis were investigated by flow cytometry, agarose gel electrophoresis, and light and electron microscopy. A subdiploid peak and DNA laddering, both of which are parameters specifically correlated to programmed cell death, were present in HL60 and U937 and, even if less evident, in DOHH₂ cells undergoing hyperthermic treatment, and were absent in K562 cells. In addition, DNA single-strand cleavage was revealed by in situ nick translation, observed by confocal microscopy. Morphological analysis confirmed these results and revealed the typical chromatin changes, followed by the appearance of micronuclei and apoptotic bodies. Accepted: 26 November 1999     

Regulation of platelet-derived growth factor gene expression by transforming growth factor beta and phorbol ester in human leukemia cell lines.

We studied the expression of the genes encoding the A and B chains of platelet-derived growth factor (PDGF) in a number of human leukemia cell lines. Steady-state expression of the A-chain RNA was seen only in the promonocytic leukemia cell line U937 and in the T-cell leukemia cell line MOLT-4. It has previously been reported that both PDGF A and PDGF B genes are induced during megakaryoblastic differentiation of the K562 erythroleukemia cells and transiently during monocytic differentiation of the promyelocytic leukemia cell line HL-60 and U937 cells. In this study we show that PDGF A RNA expression was induced in HL-60 and Jurkat T-cell leukemia cells and increased in U937 and MOLT-4 cells after a 1- to 2-h stimulation with an 8 pM concentration of transforming growth factor beta (TGF-beta). PDGF A RNA remained at a constant, elevated level for at least 24 h in U937 cells, but returned to undetectable levels within 12 h in HL-60 cells. No PDGF A expression was induced by TGF-beta in K562 cells or in lung carcinoma cells (A549). Interestingly, essentially no PDGF B-chain (c-sis proto-oncogene) RNA was expressed simultaneously with PDGF A. In the presence of TGF-beta and protein synthesis inhibitors, PDGF A RNA was superinduced at least 20-fold in the U937 and HL-60 cells. PDGF A expression was accompanied by secretion of immunoprecipitable PDGF to the culture medium of HL-60 and U937 cells. The phorbol ester tumor promoter tetradecanoyl phorbol acetate also increased PDGF A expression with similar kinetics, but with a mechanism distinct from that of TGF-beta. These results suggest a role for TGF-beta in the differential regulation of expression of the PDGF genes.     

Histone deacetylase inhibitors synergistically potentiate death receptor 4-mediated apoptotic cell death of human T-cell acute lymphoblastic leukemia cells

Cell-death signaling through the pro-apoptotic tumor necrosis factor-related apoptosis inducing ligand (TRAIL) receptors, death receptor 4 (DR4) and DR5, has shown tumor-selective apoptotic activity. Here, we examine susceptibility of various leukemia cell lines (HL-60, U937, K562, CCRF-CEM, CEM-CM3, and THP-1) to an anti-DR4 agonistic monoclonal antibody (mAb), AY4, in comparison with TRAIL. While most of the leukemia cell lines were intrinsically resistant to AY4 or TRAIL alone, the two T-cell acute lymphoblastic leukemia (T-ALL) lines, CEM-CM3 and CCRF-CEM cells, underwent synergistic caspase-dependent apoptotic cell death by combination of AY4 or TRAIL with a histone deacetylase inhibitor (HDACI), either suberoylanilide hydroxamic acid (SAHA) or valproic acid (VPA). All of the combined treatments synergistically downregulated several anti-apoptotic proteins (c-FLIP, Bcl-2, Bcl-X_L, XIAP, and survivin) without significant changing the expression levels of pro-apoptotic proteins (Bax and Bak) or the receptors (DR4 and DR5). Downregulation of c-FLIP to activate caspase-8 was a critical step for the synergistic apoptosis through both extrinsic and intrinsic apoptotic pathways. Our results demonstrate that the HDACIs have synergistic effects on DR4-specific mAb AY4-mediated cell death in the T-ALL cells with comparable competence to those exerted by TRAIL, providing a new strategy for the targeted treatment of human T-ALL cells.     

Maslinic acid induced apoptosis in bladder cancer cells through activating p38 MAPK signaling pathway

The present study determines the role of reactive oxygen species (ROS) production and calcium signaling evoked by the tumor necrosis factor-alpha (TNFα) on apoptosis in the human leukemia HL-60 and K562 cell lines. The results show that treatment of both cell lines cells with 10 ng/mL TNFα resulted in a rise in the percentage of apoptotic cells after 6 h of treatment. It was also observed that the administration of 10 ng/mL TNFα increased intracellular ROS production, as well as a time-dependent increase in caspase-8, -3, and -9 activities. The present results also show that the pretreatment with well-known antioxidants such as trolox and N-acetyl cysteine partially reduced the caspase activation caused by the administration of TNFα. The findings suggest that TNFα-induced apoptosis is dependent on alterations in intracellular ROS generation in human leukemia HL-60 and K562 cells.     

Thymosin alpha1 suppresses proliferation and induces apoptosis in human leukemia cell lines

Thymosin alpha1 (Talpha1), a 28-amino acid peptide, is a well-known immune system enhancer for the treatment of various diseases. In the present investigation, the effects of Talpha1 on the proliferation and apoptosis of human leukemia cell lines (HL-60, K562 and K562/ADM) were studied. The proliferation was significantly depressed after 96 h of treatment with Talpha1, and obvious signs of apoptosis, i.e., cell morphology, nuclei condensation and Annexin V binding, were observed thereafter. Moreover, the up-regulation of Fas/Apol (CD95) and decrease in bcl-2 anti-apoptotic gene expression were observed in apoptotic cells. The expression and the function of P-glycoprotein (P-gp) can be slightly inhibited by Talpha1. It is noteworthy that K562 and K562/ADM were more sensitive than HL-60 cells when subjected to Talpha1. Furthermore, HepG-2, the human hepatoma cell line, displayed significant less sensitivity to Talpha1 than all the human leukemia cell lines. D-Tubocurarine (TUB), a nicotinic acetylcholine receptors (nAChRs) antagonist, significantly antagonized the inhibition effects induced by Talpha1, whereas atropine, a muscarinic acetylcholine receptor antagonist, did not exhibit such effects. All the results indicate that Talpha1 was able to significantly suppress proliferation and induce apoptosis in human leukemia cell lines.     

Induction of apoptosis in K562/ADM cells by gamma-linolenic acid involves lipid peroxidation and activation of caspase-3

Numerous studies have revealed that gamma-linolenic acid (GLA) possesses effective tumoricidal properties while not inducing damage to normal cells or creating harmful systemic side effects. It can exert anti-tumor efficacy against a variety of cancers including leukemia. However, little is known about the effects of GLA on leukemia resistant to chemotherapy, emerging as a serious clinical problem. The present study tested GLA-induced apoptosis in K562/ADM multidrug-resistant (MDR) leukemic cells and investigated its possible mechanisms. Using cell viability, fluorescent staining of nuclei, flow cytometric Annexin V/PI double staining and lactate dehydrogenase (LDH) release, we found that GLA could inhibit cell growth and induce apoptosis and secondary necrosis. The results showed that incubation with GLA concentrations of 10-60 microg/ml caused a dose- and time-dependent decrease of K562/ADM cell viability, and the IC50 value was 50.5 microg/ml at 24 h and 31.5 microg/ml at 48 h. Flow cytometry using Annexin V/PI double staining assessed apoptosis, necrosis and viability. Typical apoptotic nuclei were shown by staining of K562/ADM cells with DNA-binding fluorochrome Hoechst 33342, characterized by chromatin condensation and nuclear fragmentation. On the other hand, after treated K562/ADM cells with 20 microg/ml GLA for 48 h and with 40 microg/ml GLA for 12 h, the LDH release significantly increased, indicated losses of plasma membrane integrity and presence of necrosis. Further, the inhibition of GLA-induced apoptosis by a pan-caspase inhibitor (z-VAD-fmk) suggested the involvement of caspases. The increase of caspase-3 activity with GLA concentration confirmed its role in the process. The results also showed that the malondialdehyde (MDA) content was also significantly elevated, and antioxidant BHT could block GLA cytotoxity, indicating the cytotoxity induced by GLA may be due to lipid peroxidation.     

Caspase Activation Is an Early Event in Anthracycline-Induced Apoptosis and Allows Detection of Apoptotic Cells before They Are Ingested by Phagocytes

An increasing number of methods are being described to detect apoptotic cells. However, attempts to detect apoptotic cells in clinical samples are rarely successful. A hypothesis is that apoptotic cells are cleared from the circulation by phagocytosis before they become detectable by conventional morphological or cytometric methods. Using LR73 adhering cells as phagocytes in a model ofin vitrophagocytosis, we found that phagocytosis of daunorubicin (DNR)-treated U937, HL60, or K562 leukemia cell lines occurred prior to phosphatidylserine externalization, DNA hydrolysis, chromatin condensation, nuclear fragmentation, or mitochondrial potential alteration. Moreover DNR-treated K562 cells were eliminated by phagocytes while apoptosis was never observed by any of the above methods. By contrast, using a fluorometric batch analysis assay to detect caspase activity in ceramide- or DNR-treated cells (fluorogenic substrate for caspase), we found that caspase activity increased in apoptosis-committed cells before they were detected by flow cytometry or recognized by phagocytes. Similarly a caspase activity increase was detected in circulating mononuclear cells of leukemic patients 15 h after the beginning of anthracyclin treatment. We suggest that recent findings on enzymatic events (caspase activation) occurring in the early events of apoptosis must now allow the development of new markers for apoptosis, irrespective of the morphological features or internucleosomal fragmentation which are late events in apoptosis.     

Evidence of intracellular and trans-acting differentiation-inducing activity in human promyelocytic leukemia HL-60 cells: its possible involvement in process of cell differentiation from a commitment step to a phenotype-expression step

We previously reported that human promyelocytic leukemia HL-60 cells, when treated with various inducers in magnesium-deficient medium, became committed to differentiate but did not express the differentiation-related phenotypes (Okazaki et al., J. Cell. Physiol., 131:50-57, 1987). In the present study we demonstrated the existence of an intracellular differentiation-inducing activity (int-DIA) in differentiation-committed phenotype-nonexpressing HL-60 cells by using cybrid formation between untreated HL-60 cells and cytoplasts from HL-60 cells treated in magnesium-deficient medium with 100 nM 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3). Cell extracts from similarly treated HL-60 cells also showed int-DIA, which when added (10 mg total protein/ml) to culture of untreated HL-60 cells, could increase the percentages of nitroblue tetrazolium (NBT)- and nonspecific esterase (NSE)-positive cells from 1% to 53%, and from 0 to 32%, respectively. They also induced differentiation of human monoblastic leukemia U-937 cells and of human myeloblastic leukemia KG-1 cells but not of erythroleukemia K-562 cells. These results suggested that the int-DIA had a common effect on differentiation induction in several human myeloid cell lines and may be involved in inducing cells to proceed from a commitment to a phenotype-expression step during human myeloid cell differentiation.     

Induction of differentiation and apoptosis in three human leukemia cell lines by a new compound from Dendrostellera lessertii

It has previously been shown that Dendrostellera lessertii(Thymelaeaceae)has stronganticancer activity.In this study,the antileukemic activity of another new compound from the same plantextract is reported.Promyelocytic(NB4 and HL-60)and erythroleukemia(K562)cells were cultured in thepresence of various concentrations of the new compound(0.5-3.0 μtg/ml)for 3d.The cell numbers werethen determined by trypan blue exclusion test.The new compound inhibited growth and proliferation ofNB4,HL-60 and K562 with IC_(50) values of 1.5,2.0 and 2.5μg/ml,respectively.We also found that the newcompound inhibited cell proliferation in a dose-and time-dependent manner.At low concentrations and after48h of treatment,approximately 50%-70% of NB4 and HL-60 cells were differentiated to monocyte/macrophage lineage and approximately 30%-40% of the treated K562 cells were differentiated in the mega-karyocytic lineage,as evidenced by morphological changes and nitro blue tetrazolium reduction assays.Results of Hoechst 33258 staining also indicated that the new compound induced NB4 and HL-60 cellapoptosis at their respective IC_(50) values after 72h of treatment.Based on the present data,the new com-pound seems a good candidate for further evaluation as an effective chemotherapeutic agent acting throughinduction of differentiation and apoptosis.     

Induction of HL-60 apoptosis by ethyl acetate extract of Cordyceps sinensis fungal mycelium

The cultivated mycelium of a Cordyceps sinensis (Cs) fungus was sequentially extracted by petroleum ether, ethyl acetate (EtOAc), ethanol and water. The EtOAc extract showed the most potent cytotoxic effect against the proliferation of human premyelocytic leukemia cell HL-60, with an ED50 < or = 25 microg/ml for 2-day treatment. The EtOAc extract induced the characteristic apoptotic symptoms in the HL-60 cells, DNA fragmentation and chromatin condensation, occurring within 6-8 h of treatment at a dose of 200 microg/ml. The activation of caspase-3 and the specific proteolytic cleavage of poly ADP-ribose polymerase were detected during the course of apoptosis induction. These results suggest that the Cs mycelium extract inhibited the cancer cell proliferation by inducing cell apoptosis.     

Differential Sensitivity of Telomerase from Human Hematopoietic Stem Cells and Leukemic Cell Lines to Mild Hyperthermia

We have investigated the effects of hyperthermia (HT) on cell proliferation and telomerase activity of human hematopoietic stem cells (HSCs) and compared with human leukemic cell lines (TF-1, K562 and HL-60). The cells were exposed to HT at 42 and 43 °C up to 120 min. The cells were incubated at 37 °C for 96 h. Then the cells were collected and assayed for cell proliferation, viability, telomerase activity, and terminal restriction fragment (TRF) lengths. The enzyme activity from HSCs was decreased up to 68.6 at 42 and 85.1 % at 43 °C for 120 min. This inhibition in leukemic cells was up to 28.9 and 53.6 % in TF-1; 53 and 63.9 % in K562; 45.2 and 61.1 % in HL-60 cells. The treated cells showed TRF lengths about 5.3 kb for control HL-60 cells, 5.0 kb for HL-60 cells treated at 42 and 4.5 kb at 43 °C for 120 min. In HSCs, the TRF length was about 4.5 kb for untreated cells and 4.0–4.5 kb for treated cells at 42 and 43 °C for 120 min. The time response curves indicated that, inhibition of the enzyme activity in leukemic cells was dependent to the time of exposure to HT. But in HSCs, the inhibition was reached to steady state at 15 min exposure to 43 °C heat stress. TRF length was constant at treated two types of cells, which implies that in cells subjected to mild HT no telomere shortening was observed.     

Synchronized onset of nuclear and cell surface modifications in U937 cells during apoptosis

In this study we investigated the relationship between nuclear and cell surface modifications (i.e. blebbing, phosphatidylserine [PS] and sugar residues exposure) in a monocytic cell line, U937, during apoptosis induced by oxidative stress (1 mM H2O2) or inhibition of protein synthesis (10 microg/ml puromycin). Dying cells were simultaneously observed for nuclear modifications, presence of superficial blebs and plasma membrane alterations. Morphological analysis performed by conventional fluorescence microscopy, or by transmission and scanning electron microscopy showed that the courses of nuclear and membrane alterations occured concomitantly, but the phenotype was dependent on the stage of the apoptotic process and the type of apoptogenic inducer used. The progression of apoptosis in U937 cells beyond early stages resulted in the extensive formation of blebs which concomitantly lost some typical markers of apoptosis, such as PS and sugar residues. Therefore, the modality by which the nucleus condenses, or the amount and the pattern of distribution of PS on the cell surface were, for each cell line, strictly related to the apoptogenic inducer. The morphological data reported in the present paper should lead to a more precise quantification of apoptosis by improving the detection of apoptotic cells in vivo (i.e. in tissue, organs), which is a crucial point in the evaluation of efficiency of antiproliferative drugs, such as antiblastic or immunosuppressive compounds.     

Apoptosis induction in human leukemic cells by a novel protein Bengalin,isolated from Indian black scorpion venom: Through mitochondrial pathway and inhibition of heat shock proteins

Scorpion venom possesses protein toxins having numerous biological activities, some of which are potentially anticancerous. Previously we had reported antiproliferative activity of the venom of Indian black scorpion, Heterometrus bengalensis Koch. Here we have isolated and purified a novel protein named Bengalin (72 kDa) from the venom, responsible for antiproliferative and apoptogenic activities against human leukemic cells U937 (histiocytic lymphoma) and K562 (chronic myelogenous leukemia). N-terminal sequence of first 20 amino acids of Bengalin was G-P-L-T-I-L-H-I-N-D-V-H-A-A/R-F-E-Q/G-F/G-N-T. Bengalin induced cell growth inhibition at IC₅₀ values of 3.7 and 4.1 μg/ml for U937 and K562 cells respectively did not significantly affect normal human lymphocytes. Inhibition of U937 and K562 cell proliferation occurred by apoptosis as evidenced from damaged nuclei, cell cycle arrest at sub G1 phase, increase of early apoptotic cells, augmentation of DNA fragmentation and also a reduction of telomerase activity. Further insights revealed that Bax:Bcl2 ratio was elevated after Bengalin treatment. Moreover Bengalin elicited loss of mitochondrial membrane potential (MMP) which commenced cytochrome c release in cytosol, decreased heat shock protein (HSP) 70 and 90 expression, activated caspase-9, caspase-3 and induced poly(ADP-ribose) polymerase (PARP) cleavage. We have also determined that HSP70 and 90 inhibitions correlated with Bengalin induced antiproliferation, caspase-3 upregulation, apoptogenesis and increased DNA fragmentation. These results hypothesize that Bengalin might provide a putative molecular mechanism for their anticancer effect on human leukemic cells which might be mediated by mitochondrial death cascade. Inhibition of HSPs might also play a crucial role in induction of apoptosis.