Spatiotemporal immune atlas of a clinical-grade gene-edited pig-to-human kidney xenotransplant.

Pig-to-human xenotransplantation is rapidly approaching the clinical arena; however, it is unclear which immunomodulatory regimens will effectively control human immune responses to pig xenografts. Here, we transplant a gene-edited pig kidney into a brain-dead human recipient on pharmacologic immunosuppression and study the human immune response to the xenograft using spatial transcriptomics and single-cell RNA sequencing. Human immune cells are uncommon in the porcine kidney cortex early after xenotransplantation and consist of primarily myeloid cells. Both the porcine resident macrophages and human infiltrating macrophages express genes consistent with an alternatively activated, anti-inflammatory phenotype. No significant infiltration of human B or T cells into the porcine kidney xenograft is detectable. Altogether, these findings provide proof of concept that conventional pharmacologic immunosuppression may be able to restrict infiltration of human immune cells into the xenograft early after compatible pig-to-human kidney xenotransplantation.

Spatiotemporal immune atlas of the first clinical-grade, gene-edited pig-to-human kidney xenotransplant.

Pig-to-human xenotransplantation is rapidly approaching the clinical arena; however, it is unclear which immunomodulatory regimens will effectively control human immune responses to pig xenografts. We transplanted a gene-edited pig kidney into a brain-dead human recipient on pharmacologic immunosuppression and studied the human immune response to the xenograft using spatial transcriptomics and single-cell RNA sequencing. Human immune cells were uncommon in the porcine kidney cortex early after xenotransplantation and consisted of primarily myeloid cells. Both the porcine resident macrophages and human infiltrating macrophages expressed genes consistent with an alternatively activated, anti-inflammatory phenotype. No significant infiltration of human B or T cells into the porcine kidney xenograft was detected. Altogether, these findings provide proof of concept that conventional pharmacologic immunosuppression is sufficient to restrict infiltration of human immune cells into the xenograft early after compatible pig-to-human kidney xenotransplantation.

Optimization of methods for the accurate characterization of whole blood neutrophils.

Neutrophils are the most abundant circulating leukocyte population with critical roles in immune defense, regulation of innate and adaptive immune systems, and disease pathogenesis. Our progress in understanding precise mechanisms of neutrophil activation, recruitment, and function has been hampered by the lack of optimized and standardized methods for the characterization and phenotyping of this readily activated population. By comparing eight methods of neutrophil characterization, we demonstrate that the level of neutrophil activation and degranulation is associated with specific experimental conditions and the number and type of manipulation steps employed. Staining whole blood at 4 °C and removal of remaining unbound antibodies prior to one-step fixation and red blood cell lysis minimizes neutrophil activation, decreases phenotypic alterations during processing, and prevents nonspecific antibody binding. The effects of anticoagulants used for collection, processing delays, and time and temperature during sample analysis on neutrophil phenotype are addressed. The presented data provide a foundation for higher quality standards of neutrophil characterization improving consistency and reproducibility among studies.

Distinct phenotype of neutrophil, monocyte, and eosinophil populations indicates altered myelopoiesis in a subset of patients with multiple myeloma.

Hematologic malignancies, including multiple myeloma (MM), promote systemic immune dysregulation resulting in an alteration and increased plasticity of myeloid cell subsets. To determine the heterogeneity of the myeloid cell compartment in the peripheral blood of patients with MM, we performed a detailed investigation of the phenotype and function of myeloid subpopulations. We report that a subset of MM patients exhibits a specific myeloid cell phenotype indicative of altered myelopoiesis characterized by significant changes in the properties of circulating granulocytic, monocytic, and eosinophilic populations. The subset, referred to as MM2, is defined by a markedly elevated level of CD64 (FcγRI) on the surface of circulating neutrophils. Compared to healthy controls or MM1 patients displaying intermediate levels of CD64, neutrophils from MM2 patients exhibit a less differentiated phenotype, low levels of CD10 and CXC chemokine receptor 2 (CXCR2), increased capacity for the production of mitochondrial reactive oxygen species, and an expansion of CD16neg immature neutrophil subset. Classical and patrolling monocytes from MM2 patients express elevated levels of CD64 and activation markers. MM2 eosinophils display lower levels of C-C Chemokine receptor 3 (CCR3), Toll-like receptor 4 (TLR4, CD284), and tissue factor (TF, CD142). The MM2 (CD64high) phenotype is independent of age, race, sex, and treatment type. Characteristic features of the MM2 (CD64high) phenotype are associated with myeloma-defining events including elevated involved/uninvolved immunoglobulin free light chain (FLC) ratio at diagnosis. Detailed characterization of the altered myeloid phenotype in multiple myeloma will likely facilitate the identification of patients with an increased risk of disease progression and open new avenues for the rational design of novel therapeutic approaches.

Cryptolepine inhibits melanoma cell growth through coordinated changes in mitochondrial biogenesis, dynamics and metabolic tumor suppressor AMPKα1/2-LKB1.

Dysregulated mitochondrial dynamics and biogenesis have been associated with various pathological conditions including cancers. Here, we assessed the therapeutic effect of cryptolepine, a pharmacologically active alkaloid derived from the roots of Cryptolepis sanguinolenta, on melanoma cell growth. Treatment of human melanoma cell lines (A375, Hs294t, SK-Mel28 and SK-Mel119) with cryptolepine (1.0, 2.5, 5.0 and 7.5 µM) for 24 and 48 h significantly (P < 0.001) inhibited the growth of melanoma cells but not normal melanocytes. The inhibitory effect of cryptolepine was associated with loss of mitochondrial membrane potential and reduced protein expression of Mfn1, Mfn2, Opa1 and p-Drp1 leading to disruption of mitochondrial dynamics. A decrease in the levels of ATP and mitochondrial mass were associated with activation of the metabolic tumor suppressor AMPKα1/2-LKB1, and a reduction in mTOR signaling. Decreased expression of SDH-A and COX-I demonstrated that cryptolepine treatment reduced mitochondrial biogenesis. In vivo treatment of A375 xenograft-bearing nude mice with cryptolepine (10 mg/Kg body weight, i.p.) resulted in significant inhibition of tumor growth, which was associated with disruption of mitochondrial dynamics and a reduction in mitochondrial biogenesis. Our study suggests that low toxicity phytochemicals like cryptolepine may be tested for the treatment of melanoma.

Dietary proanthocyanidins prevent ultraviolet radiation-induced non-melanoma skin cancer through enhanced repair of damaged DNA-dependent activation of immune sensitivity.

Numerous plant products have been used to prevent and manage a wide variety of diseases for centuries. These products are now considered as promising options for the development of more effective and less toxic alternatives to the systems of medicine developed primarily in developed countries in the modern era. Grape seed proanthocyanidins (GSPs) are of great interest due to their anti-carcinogenic effects that have been demonstrated using various tumor models including ultraviolet (UV) radiation-induced non-melanoma skin cancer. In a pre-clinical mouse model supplementation of a control diet (AIN76A) with GSPs at concentrations of 0.2% and 0.5% (w/w) significantly inhibits the growth and multiplicity of UVB radiation-induced skin tumors. In this review, we summarize the evidence that this inhibition of UVB-induced skin tumor development by dietary GSPs is mediated by a multiplicity of coordinated effects including: (i) Promotion of the repair of damaged DNA by nuclear excision repair mechanisms, and (ii) DNA repair-dependent stimulation of the immune system following the functional activation of dendritic cells and effector T cells. Dietary GSPs hold promise for the development of an effective alternative strategy for the prevention of excessive solar UVB radiation exposure-induced skin diseases including the risk of non-melanoma skin cancer in humans.

Cryptolepine, a Plant Alkaloid, Inhibits the Growth of Non-Melanoma Skin Cancer Cells through Inhibition of Topoisomerase and Induction of DNA Damage.

Topoisomerases have been shown to have roles in cancer progression. Here, we have examined the effect of cryptolepine, a plant alkaloid, on the growth of human non-melanoma skin cancer cells (NMSCC) and underlying mechanism of action. For this purpose SCC-13 and A431 cell lines were used as an in vitro model. Our study reveals that SCC-13 and A431 cells express higher levels as well as activity of topoisomerase (Topo I and Topo II) compared with normal human epidermal keratinocytes. Treatment of NMSCC with cryptolepine (2.5, 5.0 and 7.5 µM) for 24 h resulted in marked decrease in topoisomerase activity, which was associated with substantial DNA damage as detected by the comet assay. Cryptolepine induced DNA damage resulted in: (i) an increase in the phosphorylation of ATM/ATR, BRCA1, Chk1/Chk2 and γH2AX; (ii) activation of p53 signaling cascade, including enhanced protein expressions of p16 and p21; (iii) downregulation of cyclin-dependent kinases, cyclin D1, cyclin A, cyclin E and proteins involved in cell division (e.g., Cdc25a and Cdc25b) leading to cell cycle arrest at S-phase; and (iv) mitochondrial membrane potential was disrupted and cytochrome c released. These changes in NMSCC by cryptolepine resulted in significant reduction in cell viability, colony formation and increase in apoptotic cell death.

Fisetin and Its Role in Chronic Diseases.

Chronic inflammation is a prolonged and dysregulated immune response leading to a wide variety of physiological and pathological conditions such as neurological abnormalities, cardiovascular diseases, diabetes, obesity, pulmonary diseases, immunological diseases, cancers, and other life-threatening conditions. Therefore, inhibition of persistent inflammation will reduce the risk of inflammation-associated chronic diseases. Inflammation-related chronic diseases require chronic treatment without side effects. Use of traditional medicines and restricted diet has been utilized by mankind for ages to prevent or treat several chronic diseases. Bioactive dietary agents or "Nutraceuticals" present in several fruits, vegetables, legumes, cereals, fibers, and certain spices have shown potential to inhibit or reverse the inflammatory responses and several chronic diseases related to chronic inflammation. Due to safe, nontoxic, and preventive benefits, the use of nutraceuticals as dietary supplements or functional foods has increased in the Western world. Fisetin (3,3',4',7-tetrahydroxyflavone) is a dietary flavonoid found in various fruits (strawberries, apples, mangoes, persimmons, kiwis, and grapes), vegetables (tomatoes, onions, and cucumbers), nuts, and wine that has shown strong anti-inflammatory, anti-oxidant, anti-tumorigenic, anti-invasive, anti-angiogenic, anti-diabetic, neuroprotective, and cardioprotective effects in cell culture and in animal models relevant to human diseases. In this chapter, we discuss the beneficial pharmacological effects of fisetin against different pathological conditions with special emphasis on diseases related to chronic inflammatory conditions.

Fisetin, a dietary flavonoid, augments the anti-invasive and anti-metastatic potential of sorafenib in melanoma.

Melanoma is the most aggressive and deadly form of cutaneous neoplasm due to its propensity to metastasize. Oncogenic BRAF drives sustained activation of the BRAF/MEK/ERK (MAPK) pathway and cooperates with PI3K/AKT/mTOR (PI3K) signaling to induce epithelial to mesenchymal transition (EMT), leading to cell invasion and metastasis. Therefore, targeting these pathways is a promising preventive/therapeutic strategy. We have shown that fisetin, a flavonoid, reduces human melanoma cell invasion by inhibiting EMT. In addition, fisetin inhibited melanoma cell proliferation and tumor growth by downregulating the PI3K pathway. In this investigation, we aimed to determine whether fisetin can potentiate the anti-invasive and anti-metastatic effects of sorafenib in BRAF-mutated melanoma. We found that combination treatment (fisetin + sorafenib) more effectively reduced the migration and invasion of BRAF-mutated melanoma cells both in vitro and in raft cultures compared to individual agents. Combination treatment also effectively inhibited EMT as observed by a decrease in N-cadherin, vimentin and fibronectin and an increase in E-cadherin both in vitro and in xenograft tumors. Furthermore, combination therapy effectively inhibited Snail1, Twist1, Slug and ZEB1 protein expression compared to monotherapy. The expression of MMP-2 and MMP-9 in xenograft tumors was further reduced in combination treatment compared to individual agents. Bioluminescent imaging of athymic mice, intravenously injected with stably transfected CMV-luciferase-ires-puromycin.T2A.EGFP-tagged A375 melanoma cells, demonstrated fewer lung metastases following combination treatment versus monotherapy. Our findings demonstrate that fisetin potentiates the anti-invasive and anti-metastatic effects of sorafenib. Our data suggest that fisetin may be a worthy adjuvant chemotherapy for the management of melanoma.

Prodifferentiation, anti-inflammatory and antiproliferative effects of delphinidin, a dietary anthocyanidin, in a full-thickness three-dimensional reconstituted human skin model of psoriasis.

BACKGROUND: Psoriasis is a chronic inflammatory disorder of skin and joints for which conventional treatments that are effective in clearing the moderate-to-severe disease are limited due to long-term safety issues. This necessitates exploring the usefulness of botanical agents for treating psoriasis. We previously showed that delphinidin, a diet-derived anthocyanidin endowed with antioxidant and anti-inflammatory properties, induces normal epidermal keratinocyte differentiation and suggested its possible usefulness for the treatment of psoriasis [1]. OBJECTIVES: To investigate the effect of delphinidin (0-20 µM; 2-5 days) on psoriatic epidermal keratinocyte differentiation, proliferation and inflammation using a three-dimensional reconstructed human psoriatic skin equivalent (PSE) model. METHODS: PSEs and normal skin equivalents (NSEs) established on fibroblast-contracted collagen gels with respective psoriatic and normal keratinocytes and treated with/without delphinidin were analyzed for histology, expression of markers of differentiation, proliferation and inflammation using histomorphometry, immunoblotting, immunochemistry, qPCR and cultured supernatants for cytokine with a Multi-Analyte ELISArray Kit. RESULTS: Our data show that treatment of PSE with delphinidin induced (1) cornification without affecting apoptosis and (2) the mRNA and protein expression of markers of differentiation (caspase-14, filaggrin, loricrin, involucrin). It also decreased the expression of markers of proliferation (Ki67 and proliferating cell nuclear antigen) and inflammation (inducible nitric oxide synthase and antimicrobial peptides S100A7-psoriasin and S100A15-koebnerisin, which are often induced in psoriatic skin). ELISArray showed increased release of psoriasis-associated keratinocyte-derived proinflammatory cytokines in supernatants of the PSE cultures, and this increase was significantly suppressed by delphinidin. CONCLUSIONS: These observations provide a rationale for developing delphinidin for the management of psoriasis.

Fisetin inhibits growth, induces G2 /M arrest and apoptosis of human epidermoid carcinoma A431 cells: role of mitochondrial membrane potential disruption and consequent caspases activation.

Non-melanoma skin cancers (NMSCs), one of the most common neoplasms, cause serious morbidity and mortality. Therefore, identification of non-toxic phytochemicals for prevention/treatment of NMSCs is highly desirable. Fisetin (3,3',4',7-tetrahydroxyflavone), a dietary flavonoid, present in fruits and vegetables possesses anti-oxidant and antiproliferative properties. The aim of this study was to investigate the chemotherapeutic potential of fisetin in cultured human epidermoid carcinoma A431 cells. Treatment of A431 cells with fisetin (5-80 µm) resulted in a significant decrease in cell viability in a dose- and time-dependent manner. Employing clonogenic assay, we found that fisetin treatment significantly reduced colony formation in A431 cells. Fisetin treatment of A431 cells resulted in G2 /M arrest and induction of apoptosis. Furthermore, treatment of A431 cells with fisetin resulted in (i) decreased expression of anti-apoptotic proteins (Bcl2; Bcl-xL and Mcl-1); (ii) increased expression of pro-apoptotic proteins (Bax, Bak and Bad); (iii) disruption of mitochondrial potential; (iv) release of cytochrome c and Smac/DIABLO from mitochondria; (v) activation of caspases; and (vi) cleavage of Poly(ADP-ribose) polymerase (PARP) protein. Pretreatment of A431 cells with the pan-caspase inhibitor (Z-VAD-FMK) blocked fisetin-induced cleavage of caspases and PARP. Taken together, these data provide evidence that fisetin possesses chemotherapeutic potential against human epidermoid carcinoma A431 cells. Overall, these results suggest that fisetin could be developed as a novel therapeutic agent for the management of NMSCs.

Estrogen-anchored pH-sensitive liposomes as nanomodule designed for site-specific delivery of doxorubicin in breast cancer therapy.

The present investigation reports the development of nanoengineered estrogen receptor (ER) targeted pH-sensitive liposome for the site-specific intracellular delivery of doxorubicin (DOX) for breast cancer therapy. Estrone, a bioligand, was anchored on the surface of pH-sensitive liposome for drug targeting to ERs. The estrone-anchored pH-sensitive liposomes (ES-pH-sensitive-SL) showed fusogenic potential at acidic pH (5.5). In vitro cytotoxicity studies carried out on ER-positive MCF-7 breast carcinoma cells revealed that ES-pH-sensitive-SL formulation was more cytotoxic than non-pH-sensitive targeted liposomes (ES-SL). The flow cytometry analysis confirmed significant enhanced uptake (p < 0.05) of ES-pH-sensitive-SL by MCF-7 cells. Intracellular delivery and nuclear localization of the DOX was confirmed by fluorescence microscopy. The mechanism for higher cytotoxicity shown by estrone-anchored pH-sensitive liposomal-DOX was elucidated using reactive oxygen species (ROS) determination. The in vivo biodistribution studies and antitumor activities of formulations were evaluated on tumor bearing female Balb/c mice followed by intravenous administration. The ES-pH-sensitive-SL efficiently suppressed the breast tumor growth in comparison to both ES-SL and free DOX. Serum enzyme activities such as LDH and CPK levels were assayed for the evaluation of DOX induced cardiotoxicity. The ES-pH-sensitive-SL accelerated the intracellular trafficking of encapsulated DOX, thus increasing the therapeutic efficacy. The findings support that estrone-anchored pH-sensitive liposomes could be one of the promising nanocarriers for the targeted intracellular delivery of anticancer agents to breast cancer with reduced systemic side effects.

A novel parthenin analog exhibits anti-cancer activity: activation of apoptotic signaling events through robust NO formation in human leukemia HL-60 cells.

This study describes the anti-cancer activity of P19, an analog of parthenin. P19 induced apoptosis in HL-60 cells and inhibited cell proliferation with 48h IC50 of 3.5µM. At 10mg/kg dose, it doubled the median survival time of L1210 leukemic mice and at 25mg/kg it inhibited Ehrlich ascites tumor growth by 60%. Investigation of the mechanism of P19 induced apoptosis in HL-60 cells revealed that N-acetyl-l-cysteine (NAC) and s-methylisothiourea (sMIT) could reverse several molecular events that lead to cell death by inhibiting nitric oxide (NO) formation. It selectively produced massive NO in cells while quenching the basal ROS levels with concurrent elevation of GSH. P19 disrupted mitochondrial integrity leading to cytochrome c release and caspase-9 activation. P19 also caused caspase-8 activation by selectively elevating the expression of DR4 and DR5. All these events lead to the activation of caspase-3 leading to PARP-1 cleavage and DNA fragmentation. However, knocking down of AIF by siRNA also suppressed the apoptosis substantially thus indicating caspase independent apoptosis, too. Further, contrary to enhanced iNOS expression, its transcription factor, NF-κB (p65) was cleaved with a simultaneous increase in cytosolic IκB-alpha. In addition, P19 potently inhibited pro-survival proteins pSTAT3 and survivin. The multi-modal pro-apoptotic activity of P19 raises its potential usefulness as a promising anti-cancer therapeutic.

Effect of honey and eugenol on Ehrlich ascites and solid carcinoma.

Ehrlich ascites carcinoma is a spontaneous murine mammary adenocarcinoma adapted to ascites form and carried in outbred mice by serial intraperitoneal (i/p) passages. The previous work from our laboratory showed that honey having higher phenolic content was potent in inhibiting colon cancer cell proliferation. In this work, we extended our research to screen the antitumor activity of two selected honey samples and eugenol (one of the phenolic constituents of honey) against murine Ehrlich ascites and solid carcinoma models. Honey containing higher phenolic content was found to significantly inhibit the growth of Ehrlich ascites carcinoma as compared to other samples. When honey containing higher phenolic content was given at 25% (volume/volume) intraperitoneally (i/p), the maximum tumor growth inhibition was found to be 39.98%. However, honey was found to be less potent in inhibiting the growth of Ehrlich solid carcinoma. On the other hand, eugenol at a dose of 100 mg/kg i/p was able to inhibit the growth of Ehrlich ascites by 28.88%. In case of solid carcinoma, eugenol (100 mg/kg; i/p) showed 24.35% tumor growth inhibition. This work will promote the development of honey and eugenol as promising candidates in cancer chemoprevention.

Immune modulation and apoptosis induction: Two sides of antitumoural activity of a standardised herbal formulation of Withania somnifera.

Deregulated apoptosis and suppressed tumour reactive immunity render tumour cells to grow amok in the host body. Traditionally used botanicals may offer potential anticancer chemo-immunotherapeutic leads. We report in this study a chemically standardised herbal formulation (WSF) of Withania somnifera possessing anticancer and Th1 immune up-regulatory activities. WSF produced cytotoxicity in a panel of human cancer cell lines in vitro. The molecular mechanism of cell cytotoxicity, IC(50) 48h approximately 20mug/ml, was investigated in HL-60, where it induced apoptosis by activating both intrinsic and extrinsic signalling pathways. It induced early generation of reactive nitrogen and oxygen species (RNOS), thus producing oxidative stress mediated mitochondrial membrane potential (MMP) loss leading to the release of cytochrome c, the translocation of Bax to mitochondria and apoptosis-inducing factor to the nuclei. These events paralleled the activation of caspase-9, -3 and PARP cleavage. WSF also activated caspase-8 through enhanced expression of TNF-R1 and DR-4, suggesting also the involvement of extrinsic pathway of apoptosis. WSF at 150mg/kg, i.p., inhibited >50% tumour growth in the mouse tumour models. In tumour-bearing mice, WSF inhibited the expression of pStat-3, with a selective stimulation of Th1 immunity as evidenced by enhanced secretion of IFN-gamma and IL-2. In parallel, it enhanced the proliferation of CD4(+)/CD8(+) and NK cells along with an increased expression of CD40/CD40L/CD80. In addition, WSF also enhanced T cell activation in camptothecin treated tumour-bearing mice. WSF being safe when given orally up to 1500mg/kg to rats for 6 months may be found useful in the management of malignancy by targeting at multiple pathways.

Anticancer activity of an essential oil from Cymbopogon flexuosus.

The essential oil from a lemon grass variety of Cymbopogon flexuosus was studied for its in vitro cytotoxicity against twelve human cancer cell lines. The in vivo anticancer activity of the oil was also studied using both solid and ascitic Ehrlich and Sarcoma-180 tumor models in mice. In addition, the morphological changes in tumor cells were studied to ascertain the mechanism of cell death. The in vitro cytotoxicity studies showed dose-dependent effects against various human cancer cell lines. The IC(50) values of oil ranged from 4.2 to 79 microg/ml depending upon the cell line. In 502713 (colon) and IMR-32 (neuroblastoma) cell lines, the oil showed highest cytotoxicity with IC(50) value of 4.2 and 4.7 microg/ml, respectively. Intra-peritoneal administration of the oil significantly inhibited both ascitic and solid forms of Ehrlich and Sarcoma-180 tumors in a dose-dependent manner. The tumor growth inhibition at 200 mg/kg (i.p.) of the oil observed with both ascitic and solid tumor forms of Ehrlich Ascites carcinoma was 97.34 and 57.83 respectively. In case of Sarcoma-180, the growth inhibition at similar dose of oil was 94.07 and 36.97% in ascitic and solid forms respectively. Morphological studies of the oil treated HL-60 cells revealed loss of surface projections, chromatin condensation and apoptosis. The mitochondria showed apparent loss of cristae in the cells undergoing apoptosis. The morphological studies of Sarcoma-180 solid tumor cells from animals treated with the oil revealed condensation and fragmentation of nuclei typical of apoptosis. Morphological studies of ascites cells from animals treated with the oil too revealed the changes typical of apoptosis. Our results indicate that the oil has a promising anticancer activity and causes loss in tumor cell viability by activating the apoptotic process as identified by electron microscopy.

Induction of mitochondrial-dependent apoptosis by an essential oil from Tanacetum gracile.

The essential oil of Tanacetum gracile (Accession no. AT-01 termed AT-01 in the manuscript), a cold desert alpine highly aromatic herb, has 40 constituents including lavendulol (21.5 %), lavendulol acetate (1.7 %), alpha-pinene (11.2 %), 1,8-cineole (15.2 %), CIS-beta-ocimene (6.9 %), borneol (6.1 %), limonene (5.1 %) and chamazulene (3.7 %). AT-01 was evaluated for its anticancer activity. It inhibited HL-60 cell proliferation with an IC (50) of 27 microg/mL. Furthermore, AT-01 induced apoptosis in human leukemia HL-60 cells as measured by several biological end points. AT-01 induced apoptotic body formation, enhanced annexinV-FITC binding of the cells, increased sub-G (0) DNA fraction, loss of mitochondrial membrane potential (Deltapsi (mt)) and release of cytochrome c from mitochondria, activated caspase-9 as well as caspase-3, and increased cleavage of PARP in HL-60 cells. Thus, AT-01 induced apoptosis through the mitochondrial dependent pathway in HL-60 cells.