Asparagine transport through SLC1A5/ASCT2 and SLC38A5/SNAT5 is essential for BCP-ALL cell survival and a potential therapeutic target.

B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) blasts strictly depend on the transport of extra-cellular asparagine (Asn), yielding a rationale for L-asparaginase (ASNase) therapy. However, the carriers used by ALL blasts for Asn transport have not been identified yet. Exploiting RS4;11 cells as BCP-ALL model, we have found that cell Asn is lowered by either silencing or inhibition of the transporters ASCT2 or SNAT5. The inhibitors V-9302 (for ASCT2) and GluγHA (for SNAT5) markedly lower cell proliferation and, when used together, suppress mTOR activity, induce autophagy and cause a severe nutritional stress, leading to a proliferative arrest and a massive cell death in both the ASNase-sensitive RS4;11 cells and the relatively ASNase-insensitive NALM-6 cells. The cytotoxic effect is not prevented by coculturing leukaemic cells with primary mesenchymal stromal cells. Leukaemic blasts of paediatric ALL patients express ASCT2 and SNAT5 at diagnosis and undergo marked cytotoxicity when exposed to the inhibitors. ASCT2 expression is positively correlated with the minimal residual disease at the end of the induction therapy. In conclusion, ASCT2 and SNAT5 are the carriers exploited by ALL cells to transport Asn, and ASCT2 expression is associated with a lower therapeutic response. ASCT2 may thus represent a novel therapeutic target in BCP-ALL.

Tracking fibrosis in myeloproliferative neoplasms by CCR2 expression on CD34+ cells.

In myeloproliferative neoplasm (MPNs), bone marrow fibrosis - mainly driven by the neoplastic megakaryocytic clone - dictates a more severe disease stage with dismal prognosis and higher risk of leukemic evolution. Therefore, accurate patient allocation into different disease categories and timely identification of fibrotic transformation are mandatory for adequate treatment planning. Diagnostic strategy still mainly relies on clinical/laboratory assessment and bone marrow histopathology, which, however, requires an invasive procedure and frequently poses challenges also to expert hemopathologists. Here we tested the diagnostic accuracy of the detection, by flow cytometry, of CCR2+CD34+ cells to discriminate among MPN subtypes with different degrees of bone marrow fibrosis. We found that the detection of CCR2 on MPN CD34+ cells has a very good diagnostic accuracy for the differential diagnosis between "true" ET and prePMF (AUC 0.892, P<0.0001), and a good diagnostic accuracy for the differential diagnosis between prePMF and overtPMF (AUC 0.817, P=0.0089). Remarkably, in MPN population, the percentage of CCR2-expressing cells parallels the degree of bone marrow fibrosis. In ET/PV patients with a clinical picture suggestive for transition into spent phase, we demonstrated that only patients with confirmed secondary MF showed significantly higher levels of CCR2+CD34+ cells. Overall, flow cytometric CCR2+CD34+ cell detection can be envisioned in support of conventional bone marrow histopathology in compelling clinical scenarios, with the great advantage of being extremely rapid. For patients in follow-up, its role can be conceived as an initial patient screening for subsequent bone marrow biopsy when disease evolution is suspected.

Buffering Adaptive Immunity by Hydrogen Sulfide.

T cell-mediated adaptive immunity is designed to respond to non-self antigens and pathogens through the activation and proliferation of various T cell populations. T helper 1 (Th1), Th2, Th17 and Treg cells finely orchestrate cellular responses through a plethora of paracrine and autocrine stimuli that include cytokines, autacoids, and hormones. Hydrogen sulfide (H2S) is one of these mediators able to induce/inhibit immunological responses, playing a role in inflammatory and autoimmune diseases, neurological disorders, asthma, acute pancreatitis, and sepsis. Both endogenous and exogenous H2S modulate numerous important cell signaling pathways. In monocytes, polymorphonuclear, and T cells H2S impacts on activation, survival, proliferation, polarization, adhesion pathways, and modulates cytokine production and sensitivity to chemokines. Here, we offer a comprehensive review on the role of H2S as a natural buffer able to maintain over time a functional balance between Th1, Th2, Th17 and Treg immunological responses.

ALL blasts drive primary mesenchymal stromal cells to increase asparagine availability during asparaginase treatment.

Mechanisms underlying the resistance of acute lymphoblastic leukemia (ALL) blasts to l-asparaginase are still incompletely known. Here we demonstrate that human primary bone marrow mesenchymal stromal cells (MSCs) successfully adapt to l-asparaginase and markedly protect leukemic blasts from the enzyme-dependent cytotoxicity through an amino acid trade-off. ALL blasts synthesize and secrete glutamine, thus increasing extracellular glutamine availability for stromal cells. In turn, MSCs use glutamine, either synthesized through glutamine synthetase (GS) or imported, to produce asparagine, which is then extruded to sustain asparagine-auxotroph leukemic cells. GS inhibition prevents mesenchymal cells adaptation to l-asparaginase, lowers glutamine secretion by ALL blasts, and markedly hinders the protection exerted by MSCs on leukemic cells. The pro-survival amino acid exchange is hindered by the inhibition or silencing of the asparagine efflux transporter SNAT5, which is induced in mesenchymal cells by ALL blasts. Consistently, primary MSCs from ALL patients express higher levels of SNAT5 (P < .05), secrete more asparagine (P < .05), and protect leukemic blasts (P < .05) better than MSCs isolated from healthy donors. In conclusion, ALL blasts arrange a pro-leukemic amino acid trade-off with bone marrow mesenchymal cells, which depends on GS and SNAT5 and promotes leukemic cell survival during l-asparaginase treatment.

NK cells: A double edge sword against SARS-CoV-2.

Natural killer (NK) cells are pivotal effectors of the innate immunity protecting an individual from microbes. They are the first line of defense against invading viruses, given their substantial ability to directly target infected cells without the need for specific antigen presentation. By establishing cellular networks with a variety of cell types such as dendritic cells, NK cells can also amplify and modulate antiviral adaptive immune responses. In this review, we will examine the role of NK cells in SARS-COV2 infections causing the ongoing COVID19 pandemic, keeping in mind the controversial role of NK cells specifically in viral respiratory infections and in inflammatory-driven lung damage. We discuss lessons learnt from previous coronavirus outbreaks in humans (caused by SARS-CoV-1 and MERS-COV).

Sighting acute myocardial infarction through platelet gene expression.

Acute myocardial infarction is primarily due to coronary atherosclerotic plaque rupture and subsequent thrombus formation. Platelets play a key role in the genesis and progression of both atherosclerosis and thrombosis. Since platelets are anuclear cells that inherit their mRNA from megakaryocyte precursors and maintain it unchanged during their life span, gene expression profiling at the time of an acute myocardial infarction provides information concerning the platelet gene expression preceding the coronary event. In ST-segment elevation myocardial infarction (STEMI), a gene-by-gene analysis of the platelet gene expression identified five differentially expressed genes: FKBP5, S100P, SAMSN1, CLEC4E and S100A12. The logistic regression model used to combine the gene expression in a STEMI vs healthy donors score showed an AUC of 0.95. The same five differentially expressed genes were externally validated using platelet gene expression data from patients with coronary atherosclerosis but without thrombosis. Platelet gene expression profile highlights five genes able to identify STEMI patients and to discriminate them in the background of atherosclerosis. Consequently, early signals of an imminent acute myocardial infarction are likely to be found by platelet gene expression profiling before the infarction occurs.

Role of physical exercise in the regulation of epigenetic mechanisms in inflammation, cancer, neurodegenerative diseases, and aging process.

The genetic heritage for decades has been considered to respond only to gene promoters or suppressors, with specific roles for oncogenes or tumor-suppressor genes. Epigenetics is progressively attracting increasing interest because it has demonstrated the capacity of these regulatory processes to regulate the gene expression without modifying gene sequence. Several factors may influence epigenetics, such as lifestyles including food selection. A role for physical exercise is emerging in the epigenetic regulation of gene expression. In this review, we resume physiological and pathological implications of epigenetic modification induced by the physical activity (PA). Inflammation and cancer mechanisms, immune system, central nervous system, and the aging process receive benefits due to PA through epigenetic mechanisms. Thus, the modulation of epigenetic processes by physical exercise positively influences prevention, development, and the course of inflammatory and cancer diseases, as well as neurodegenerative illnesses. This growing field of studies gives rise to a new role for PA as an option in prevention strategies and to integrate pharmacological therapeutic treatments.

Protein Kinase C Epsilon Is a Key Regulator of Mitochondrial Redox Homeostasis in Acute Myeloid Leukemia.

Purpose: The intracellular redox environment of acute myeloid leukemia (AML) cells is often highly oxidized compared to healthy hematopoietic progenitors and this is purported to contribute to disease pathogenesis. However, the redox regulators that allow AML cell survival in this oxidized environment remain largely unknown.Experimental Design: Utilizing several chemical and genetically-encoded redox sensing probes across multiple human and mouse models of AML, we evaluated the role of the serine/threonine kinase PKC-epsilon (PKCε) in intracellular redox biology, cell survival and disease progression.Results: We show that RNA interference-mediated inhibition of PKCε significantly reduces patient-derived AML cell survival as well as disease onset in a genetically engineered mouse model (GEMM) of AML driven by MLL-AF9. We also show that PKCε inhibition induces multiple reactive oxygen species (ROS) and that neutralization of mitochondrial ROS with chemical antioxidants or co-expression of the mitochondrial ROS-buffering enzymes SOD2 and CAT, mitigates the anti-leukemia effects of PKCε inhibition. Moreover, direct inhibition of SOD2 increases mitochondrial ROS and significantly impedes AML progression in vivo Furthermore, we report that PKCε over-expression protects AML cells from otherwise-lethal doses of mitochondrial ROS-inducing agents. Proteomic analysis reveals that PKCε may control mitochondrial ROS by controlling the expression of regulatory proteins of redox homeostasis, electron transport chain flux, as well as outer mitochondrial membrane potential and transport.Conclusions: This study uncovers a previously unrecognized role for PKCε in supporting AML cell survival and disease progression by regulating mitochondrial ROS biology and positions mitochondrial redox regulators as potential therapeutic targets in AML. Clin Cancer Res; 24(3); 608-18. ©2017 AACR.

PKCε Controls Mitotic Progression by Regulating Centrosome Migration and Mitotic Spindle Assembly.

To form a proper mitotic spindle, centrosomes must be duplicated and driven poleward in a timely and controlled fashion. Improper timing of centrosome separation and errors in mitotic spindle assembly may lead to chromosome instability, a hallmark of cancer. Protein kinase C epsilon (PKCε) has recently emerged as a regulator of several cell-cycle processes associated with the resolution of mitotic catenation during the metaphase-anaphase transition and in regulating the abscission checkpoint. However, an engagement of PKCε in earlier (pre)mitotic events has not been addressed. Here, we now establish that PKCε controls prophase-to-metaphase progression by coordinating centrosome migration and mitotic spindle assembly in transformed cells. This control is exerted through cytoplasmic dynein function. Importantly, it is also demonstrated that the PKCε dependency of mitotic spindle organization is correlated with the nonfunctionality of the TOPO2A-dependent G2 checkpoint, a characteristic of many transformed cells. Thus, PKCε appears to become specifically engaged in a programme of controls that are required to support cell-cycle progression in transformed cells, advocating for PKCε as a potential cancer therapeutic target.Implications: The close relationship between PKCε dependency for mitotic spindle organization and the nonfunctionality of the TOPO2A-dependent G2 checkpoint, a hallmark of transformed cells, strongly suggests PKCε as a therapeutic target in cancer. Mol Cancer Res; 16(1); 3-15. ©2017 AACR.

Platelet expression of PKCepsilon oncoprotein in myelofibrosis is associated with disease severity and thrombotic risk.

BACKGROUND: Myelofibrosis (MF) is the most aggressive Philadelphia-negative chronic myeloproliferative neoplasm (MPN) with high morbidity and mortality due to thrombo-hemorrhagic complications and leukemic transformation. MF is characterized by profound alterations of megakaryocytopoiesis, with consequent abnormalities in platelet number and function. We recently showed that the overexpression of the oncoprotein PKCepsilon plays a key role in the aberrant differentiation of MF megakaryocyte clone and that its levels correlate with disease burden. Moreover, our group previously demonstrated that PKCepsilon is over-expressed in platelets from patients with acute myocardial infarction (MI) and accounts for their increased reactivity. On these bases, we investigated here the activation state and PKCepsilon expression of MF platelets, testing potential correlations with thrombotic risk and disease aggressiveness. METHODS: Platelets were isolated from peripheral blood samples of MF patients and healthy donors (HDs). Patients were stratified according to the IPSS/DIPSS risk category and history of cardiovascular events. Platelet activation was assessed by flow cytometry. PKCepsilon mRNA and protein levels were determined by real time-PCR and western blot. RESULTS: MF platelets circulate in an activated status and display significantly higher levels of PKCepsilon compared to HDs. In MF patients, PKCepsilon platelet levels were associated with high-risk disease as well as with a positive history of major cardiovascular events. CONCLUSIONS: PKCepsilon is configuring as the common denominator of neoplastic transformation and thrombus formation in MF. Overall, our data pinpoint PKCepsilon as a potential novel biomarker of disease aggressiveness and thrombotic risk in this hematologic neoplasm.

Human thrombopoiesis depends on Protein kinase Cδ/protein kinase Cε functional couple.

A deeper understanding of the molecular events driving megakaryocytopoiesis and thrombopoiesis is essential to regulate in vitro and in vivo platelet production for clinical applications. We previously documented the crucial role of PKCε in the regulation of human and mouse megakaryocyte maturation and platelet release. However, since several data show that different PKC isoforms fulfill complementary functions, we targeted PKCε and PKCδ, which show functional and phenotypical reciprocity, at the same time as boosting platelet production in vitro. Results show that PKCδ, contrary to PKCε, is persistently expressed during megakaryocytic differentiation, and a forced PKCδ down-modulation impairs megakaryocyte maturation and platelet production. PKCδ and PKCε work as a functional couple with opposite roles on thrombopoiesis, and the modulation of their balance strongly impacts platelet production. Indeed, we show an imbalance of PKCδ/PKCε ratio both in primary myelofibrosis and essential thrombocythemia, featured by impaired megakaryocyte differentiation and increased platelet production, respectively. Finally, we demonstrate that concurrent molecular targeting of both PKCδ and PKCε represents a strategy for in vitro platelet factories.

Cytofluorimetric platelet analysis.

Blood platelets are highly specialized cells that drive hemostatic events and tissue repair mechanisms at the site of vascular injury. Their peculiar morphology and certain functional characteristics can be analyzed by flow cytometry (FCM). Specifically, platelet activation, a hallmark of prothrombotic states and inflammatory conditions, is associated with changes in expression of both surface and intracellular antigens that are recognized by specific monoclonal antibodies. Assessment of platelet activation status as ex vivo or in vitro reactivity to specific agonists has become relevant in particular conditions (namely, cardiovascular diseases, hematological malignancies, monitoring of pharmacological antiaggregation). In addition, aberrant surface marker expression that characterizes inherited and acquired platelet function disorders is also detected by FCM. This review discusses the main applications of FCM in platelet analyses, which are relevant for both research and clinical settings.

A rapid method for obtaining mesenchymal stem cells and platelets from bone marrow aspirate.

Mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) are currently used alone or in combination for therapeutic applications especially for bone repair. We tested whether MSCs can be isolated from bone marrow (BM) aspirate using a commercially available kit commonly used to obtain PRP from peripheral blood (PB). Results revealed that mononuclear cells and platelets from both PB and BM could be efficiently isolated by obtaining a mononuclear and platelet rich fraction (PB-MPRF and BM-MPRF, respectively). Starting with comparable volumes, the number of platelets increased 1.5-fold in BM-MPRF compared to PB-MPRF. The number of clonogenic cells in BM-MPRF samples was significantly higher than whole BM samples as revealed by CFU-F assay (54.92 ± 8.55 CFU-F/1.5 x 10(5) nucleated cells and 32.50 ± 12.43 CFU-F/1.5 x 10(5) nucleated cells, respectively). Cells isolated from BM-MPRF after in vitro expansion fulfilled the definition of MSCs by phenotypic criteria, and differentiated along osteogenic, adipogenic and chondrogenic lineages following induction. Results showed that the kit isolated MSCs and platelets from BM aspirate. Isolated MSCs were further expanded in a laboratory and BM-MPRF was used clinically following BM withdrawal for rapid intra-operative cell therapy for the treatment of bone defects.

Toll-like receptor 4 is involved in the cell cycle modulation and required for effective human cytomegalovirus infection in THP-1 macrophages.

Suitable host cell metabolic conditions are fundamental for the effective development of the human cytomegalovirus (HCMV) lytic cycle. Indeed, several studies have demonstrated the ability of this virus to interfere with cell cycle regulation, mainly by blocking proliferating cells in G1 or G1/S. In the present study, we demonstrate that HCMV deregulates the cell cycle of THP-1 macrophages (a cell line irreversibly arrested in G0) by pushing them into S and G2 phases. Moreover, we show that HCMV infection of THP-1 macrophages leads to Toll-like receptor 4 (TLR4) activation. Since various studies have indicated TLR4 to be involved in promoting cell proliferation, here we investigate the possible role of TLR4 in the observed HCMV-induced cell cycle perturbation. Our data strongly support TLR4 as a mediator of HCMV-triggered cell cycle activation in THP-1 macrophages favouring, in turn, the development of an efficient viral lytic cycle.

Protein kinase C ε expression in platelets from patients with acute myocardial infarction.

OBJECTIVE: Platelets play crucial roles in the pathophysiology of thrombosis and myocardial infarction. Protein kinase C ε (PKCε) is virtually absent in human platelets and its expression is precisely regulated during human megakaryocytic differentiation. On the basis of what is known on the role of platelet PKCε in other species, we hypothesized that platelets from myocardial infarction patients might ectopically express PKCε with a pathophysiological role in the disease. METHODS AND RESULTS: We therefore studied platelet PKCε expression from 24 patients with myocardial infarction, 24 patients with stable coronary artery disease and 24 healthy subjects. Indeed, platelets from myocardial infarction patients expressed PKCε with a significant frequency as compared to both stable coronary artery disease and healthy subjects. PKCε returned negative during patient follow-up. The forced expression of PKCε in normal donor platelets significantly increased their response to adenosine diphosphate-induced activation and adhesion to subendothelial collagen. CONCLUSIONS: Our data suggest that platelet generations produced before the acute event retain PKCε-mRNA that is not down-regulated during terminal megakaryocyte differentiation. Results are discussed in the perspective of peri-infarctual megakaryocytopoiesis as a critical component of myocardial infarction pathophysiology.

Downregulation of A(1) and A(2B) adenosine receptors in human trisomy 21 mesenchymal cells from first-trimester chorionic villi.

Human reproduction is complex and prone to failure. Though causes of miscarriage remain unclear, adenosine, a proangiogenic nucleoside, may help determine pregnancy outcome. Although adenosine receptor (AR) expression has been characterized in euploid pregnancies, no information is available for aneuploidies, which, as prone to spontaneous abortion (SA), are a potential model for shedding light on the mechanism regulating this event. AR expression was investigated in 71 first-trimester chorionic villi (CV) samples and cultured mesenchymal cells (MC) from euploid and TR21 pregnancies, one of the most frequent autosomal aneuploidy, with a view to elucidating their potential role in the modulation of vascular endothelial growth factor (VEGF) and nitric oxide (NO). Compared to euploid cells, reduced A(1) and A(2B) expression was revealed in TR21 CV and MCs. The non-selective adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased NO, by activating, predominantly, A(1)AR and A(2A)AR through a molecular pathway involving hypoxia-inducible-factor-1 (HIF-1α), and increased VEGF, mainly through A(2B). In conclusion the adenosine transduction cascade appears to be disturbed in TR21 through reduced expression of A(2B) and A(1)ARs. These anomalies may be implicated in complications such as fetal growth restriction, malformation and/or SA, well known features of aneuploid pregnancies. Therefore A(1) and A(2B)ARs could be potential biomarkers able to provide an early indication of SA risk and their stimulation may turn out to improve fetoplacental perfusion by increasing NO and VEGF.

TRAIL up-regulation must be accompanied by a reciprocal PKCε down-regulation during differentiation of colonic epithelial cell: implications for colorectal cancer cell differentiation.

PKC isoenzymes play central roles in various cellular signalling pathways, participating in a variety of protein phosphorylation cascades that regulate/modulate cellular structure and gene expression. It has been firmly established that several isoforms of PKC have a role in the regulation of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) activity. Our interest in probing the role of the epsilon isoform of PKC in the colonic cell differentiation stems from the discovery that PKCε and TRAIL are involved in the differentiation of other cell types like hematopoietic stem cells. Although the role of PKCε and TRAIL in the gastrointestinal system is unclear, it has been observed that PKCε has oncogenic activity in colon epithelial cells (CEC), while TRAIL increases the death of intestinal epithelial cells during inflammation. Here we demonstrate a reciprocal expression of PKCε and TRAIL in human colon mucosa: CECs at the bottom of the colonic crypts show high levels of PKCε, being negative for TRAIL expression. On the contrary, luminal CECs are positive for TRAIL, while negative for PKCε. Indeed, TRAIL- and butyrate-induced differentiation of the human colorectal cancer cell line HT29 requires the decrease of PKCε expression, whose absence in turn increases cell sensitivity to TRAIL-induced apoptosis. Moreover, TRAIL preferentially promotes HT29 differentiation into goblet cells. Taken together, this data demonstrate that TRAIL and PKCε must be reciprocally regulated to ensure physiological CEC differentiation starting from the stem cell pool, and that the down-regulation of PKCε is however critical for the differentiation and apoptosis of cancer cells.

Hypoxia-induced down-modulation of PKCepsilon promotes trail-mediated apoptosis of tumor cells.

Tumor oxygen status is considered as a prognostic marker that impacts on malignant progression and outcome of tumor therapy. TNF-related apoptosis inducing ligand (TRAIL) plays a key role in cancer immunity, with potential applications in cancer therapy. Protein kinase C (PKC)epsilon, a transforming oncogene, has a role in the protection of cardiomyocytes and neurons from hypoxia-induced damage while, it can also modulate the susceptibility of tumor cells to TRAIL-induced cell death. Here we demonstrate that hypoxia induces a tumor cell phenotype highly sensitive to the cytotoxic effects of TRAIL. Based on the observation that: i) PKCepsilon expression levels are impaired during hypoxia, ii) the overexpression of PKCepsilon, but not of a kinase-inactive PKCepsilon mutant, is able to revert the hypoxia-induced sensitivity to TRAIL, iii) the down-modulation of PKCepsilon levels by RNA interference, on the contrary, induces the highly TRAIL-sensitive phenotype, iv) the inhibition of hypoxia-inducible transcription factor-1alpha (HIF-1alpha) by specific siRNA blocks both the hypoxia-induced down-modulation of PKCepsilon and the induction of the highly TRAIL-sensitive phenotype; we conclude that the HIF-1alpha upregulation during hypoxia is associated to PKCepsilon down-modulation that likely represents the key molecular event promoting the apoptogenic effects of TRAIL in hypoxic tumor cells.

A(2B) and A(3) adenosine receptors modulate vascular endothelial growth factor and interleukin-8 expression in human melanoma cells treated with etoposide and doxorubicin.

Cancer patients undergoing treatment with systemic cancer chemotherapy drugs often have abnormal growth factor and cytokine profiles. Thus, serum levels of interleukin-8 (IL-8) are elevated in patients with malignant melanoma. In addition to IL-8, aggressive melanoma cells secrete, through its transcriptional regulator hypoxia-inducible factor 1 (HIF-1), vascular endothelial growth factor (VEGF), which promotes angiogenesis and metastasis of human cancerous cells. Whether these responses are related to adenosine, a ubiquitous mediator expressed at high concentrations in cancer and implicated in numerous inflammatory processes, is not known and is the focus of this study. We have examined whether the DNA-damaging agents etoposide (VP-16) and doxorubicin can affect IL-8, VEGF, and HIF-1 expressions in human melanoma cancer cells. In particular, we have investigated whether these responses are related to the modulation of the adenosine receptor subtypes, namely, A(1), A(2A), A(2B), and A(3). We have demonstrated that A(2B) receptor blockade can impair IL-8 production, whereas blocking A(3) receptors, it is possible to further decrease VEGF secretion in melanoma cells treated with VP-16 and doxorubicin. This understanding may present the possibility of using adenosine antagonists to reduce chemotherapy-induced inflammatory cytokine production and to improve the ability of chemotherapeutic drugs to block angiogenesis. Consequently, we conclude that adenosine receptor modulation may be useful for refining the use of chemotherapeutic drugs to treat human cancer more effectively.