Kinase profiling of liposarcomas using RNAi and drug screening assays identified druggable targets.

BACKGROUND: Liposarcoma, the most common soft tissue tumor, is understudied cancer, and limited progress has been made in the treatment of metastatic disease. The Achilles heel of cancer often is their kinases that are excellent therapeutic targets. However, very limited knowledge exists of therapeutic critical kinase targets in liposarcoma that could be potentially used in disease management. METHODS: Large RNAi and small-molecule tyrosine kinase inhibitor screens were performed against the proliferative capacity of liposarcoma cell lines of different subtypes. Each small molecule inhibitor was either FDA approved or in a clinical trial. RESULTS: Screening assays identified several previously unrecognized targets including PTK2 and KIT in liposarcoma. We also observed that ponatinib, multi-targeted tyrosine kinase inhibitor, was the most effective drug with anti-growth effects against all cell lines. In vitro assays showed that ponatinib inhibited the clonogenic proliferation of liposarcoma, and this anti-growth effect was associated with apoptosis and cell cycle arrest at the G0/G1 phase as well as a decrease in the KIT signaling pathway. In addition, ponatinib inhibited in vivo growth of liposarcoma in a xenograft model. CONCLUSIONS: Two large-scale kinase screenings identified novel liposarcoma targets and a FDA-approved inhibitor, ponatinib with clear anti-liposarcoma activity highlighting its potential therapy for treatment of this deadly tumor.

Activation of protein phosphatase 2A tumor suppressor as potential treatment of pancreatic cancer.

We utilized three tiers of screening to identify novel therapeutic agents for pancreatic cancers. First, we analyzed 14 pancreatic cancer cell lines against a panel of 66 small-molecule kinase inhibitors and dasatinib was the most potent. Second, we performed RNA expression analysis on 3 dasatinib-resistant and 3 dasatinib-sensitive pancreatic cancer cell lines to profile their gene expression. Third, gene profiling data was integrated with the Connectivity Map database to search for potential drugs. Thioridazine was one of the top ranking small molecules with highly negative enrichment. Thioridazine and its family members of phenothiazine including penfluridol caused pancreatic cancer cell death and affected protein expression levels of molecules involved in cell cycle regulation, apoptosis, and multiple kinase activities. This family of drugs causes activation of protein phosphatase 2 (PP2A). The drug FTY-720 (activator of PP2A) induced apoptosis of pancreatic cancer cells. Silencing catalytic unit of PP2A rendered pancreatic cancer cells resistant to penfluridol. Our observations suggest potential therapeutic use of penfluridol or similar agent associated with activation of PP2A in pancreatic cancers.

Per2 is a C/EBP target gene implicated in myeloid leukemia.

Circadian rhythms are endogenous biological clocks that govern fundamental physiological and behavioral functions. Consequently, perturbations of these rhythms have been associated with pathogenic conditions, such as depression, diabetes, and cancer. CCAAT/enhancer-binding proteins (C/EBPs) are a family of transcription factors that regulate cell growth and differentiation in various tissues and have also been implicated in many cancer types. Using expression profiling studies, we found that the levels of 2 core components of the circadian network, Per2 and Rev-Erb alpha, are significantly altered by C/EBPs. Further studies showed that levels of Per2 were reduced in lymphoma and acute myeloid leukemia patient samples, as well as in lymphoma cell lines. Overexpression of Per2 in hematopoietic cancer cell lines resulted in growth inhibition, cell cycle arrest, apoptosis and loss of clonogenic ability. These results support the emerging role of circadian genes in tumor suppression.

A role for the clock gene per1 in prostate cancer.

Circadian rhythms regulate diverse physiologic processes, including homeostatic functions of steroid hormones and their receptors. Perturbations of these rhythms are associated with pathogenic conditions, such as depression, diabetes, and cancer. Androgens play an important role in both normal development and carcinogenesis of the prostate. In the present study, we investigated a potential role for the core clock factor Per1 in the pathogenesis of prostate cancer. Serum-shocked synchronized prostate cancer cells displayed disrupted circadian rhythms compared with the normal prostate tissue. Using Oncomine to perform a meta-analysis of microarray expression studies, we found that Per1 is down-regulated in human prostate cancer samples compared with normal prostates. Reporter assays showed that Per1 inhibited transactivation of the androgen receptor (AR) both in 293T cells overexpressing the AR and in the prostate cancer cell line LNCaP. Forced expression of Per1 in LNCaP cells diminished the expression of known androgen-sensitive genes following stimulation with dihydrotestosterone. We showed that Per1 physically interacted with AR; in addition, we found that Per1 itself is regulated by androgens in prostate cancer cells. Overexpression of Per1 in prostate cancer cells resulted in significant growth inhibition and apoptosis. Our results support the emerging role of circadian genes as key players in malignant transformation. Further elucidating the connections between clock genes and the AR pathway could benefit the development of new therapeutic strategies for prostate cancer as well as provide insights into chronotherapy as a way to optimize current therapies.

Cucurbitacin B markedly inhibits growth and rapidly affects the cytoskeleton in glioblastoma multiforme.

Glioblastoma Multiforme (GBM) is almost inevitably a fatal tumor of the brain with most individuals dying within 1 year of diagnosis. It is the most frequent brain tumor in adults. Dose-response studies showed that Cucurbitacin B inhibited 50% growth (ED(50)) of 5 human GBM cell lines in liquid culture at approximately 10(-7) M. Soft-gel assays demonstrated that nearly all of the GBM clonogenic cells were inhibited at 10(-8) M of Cucurbitacin B. FACS analysis found that the compound (10(-7) M, 24 hr) caused G2/M arrest. The GBM cells underwent profound morphologic changes within 15-30 min after exposure to Cucurbitacin B (10(-7) M), rounding up and losing their pseudopodia associated with disruption of actin and microtubules, as observed by immunoflourescence. Cucurbitacin B (10(-7) M) caused prominent multinucleation of the cells after they were pulse-exposed (48 hr) to the drug, washed and cultured in normal medium for an additional 2 days. The drug (10(-7) M, 3-24 hr) increased levels of p-p38, p-JNK and p-JUN in U87 and T98G GBM cell lines as seen by Western blot. Interestingly, alterations in cell morphology caused by Cucurbitacin B (10(-7) M) were blocked by the JNK inhibitor SP600125. In summary, Cucurbitacin B has a prominent anti-proliferative activity on GBM cells; and at least in part, the mode of action is by affecting the cytoskeleton, as well as, the JNK pathway. Clinical trails of this drug should be pursued in GBM.

Saw Palmetto induces growth arrest and apoptosis of androgen-dependent prostate cancer LNCaP cells via inactivation of STAT 3 and androgen receptor signaling.

PC-SPES is an eight-herb mixture that has an activity against prostate cancer. Recently, we purified Saw Palmetto (Serenoa repens) from PC-SPES and found that Saw Palmetto induced growth arrest of prostate cancer LNCaP, DU145, and PC3 cells with ED50s of approximately 2.0, 2.6, and 3.3 microl/ml, respectively, as measured by mitochondrial-dependent conversion of the the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Saw Palmetto induced apoptosis of LNCaP cells in a time- and dose-dependent manner as measured by TUNEL assays. Also, Saw Palmetto increased the expression of p21waf1 and p53 protein in LNCaP cells. In addition, we found that Saw Palmetto down-regulated DHT- or IL-6-induced expression of prostate specific antigen in conjunction with down-regulation of the level of androgen receptor in the nucleus as measured by Western blot analysis. Moreover, Saw Palmetto down-regulated the IL-6-induced level of the phosphorylated form of STAT 3 in LNCaP cells. Furthermore, Saw Palmetto inhibited the growth of LNCaP cells present as tumor xenografts in BALB/c nude mice without adverse effect. These results indicate that Saw Palmetto might be useful for the treatment of individuals with prostate cancer.

In vitro and in vivo evaluation and a case report of intense nanosecond pulsed electric field as a local therapy for human malignancies.

When delivered to cells, very short duration, high electric field pulses (nanoelectropulses) induce primarily intracellular events. We present evidence that this emerging modality may have a role as a local cancer therapy. Five hematologic and 16 solid tumor cell lines were pulsed in vitro. Hematologic cells proved particularly sensitive to nanoelectropulses, with more than a 60% decrease in viable cells measured by MTT assay 96 hr after pulsing in 4 of 5 cell lines. In solid tumor cell lines, 10 out of 16 cell lines had more than a 10% decrease in viable cells. AsPC-1, a pancreatic cancer cell line, demonstrated the greatest in vitro sensitivity among solid tumor cell lines, with a 64% decrease in viable cells. When nanoelectropulse therapy was applied to AsPC-1 tumors in athymic nude mice, responses were seen in 4 of 6 tumors, including clinical complete responses in 3 of 6 animals. A single human subject applied nanoelectropulse therapy to his own basal cell carcinoma and had a complete pathologic response. In summary, we demonstrate that electric pulses 20 ns or less kill a wide variety of human cancer cells in vitro, induce tumor regression in vivo, and show efficacy in a single human patient. Therefore, nanoelectropulse therapy deserves further study as a potentially effective cancer therapy.

Scutellaria baicalensis, a herbal medicine: anti-proliferative and apoptotic activity against acute lymphocytic leukemia, lymphoma and myeloma cell lines.

Scutellaria baicalensis (S.B.) is a widely used Chinese herbal medicine. We initially investigated its in vitro anti-tumor activities. S.B inhibited the growth of ALL, lymphoma and myeloma cell lines by inducing apoptosis and cell cycle arrest at clinically achievable concentrations. The anti-proliferative effect was associated with mitochondrial damage, modulation of the Bcl family of genes, increased level of the CDK inhibitor p27(KIP1) and decreased level of c-myc oncogene. HPLC analysis of S.B. showed it contains 21% baicalin and further studies confirmed it was the major anti-cancer component of S.B. Thus, Scutellaria baicalensis should be tested in clinical trials for these hematopoietic malignancies.

PC-SPES down-regulates COX-2 via inhibition of NF-kappaB and C/EBPbeta in non-small cell lung cancer cells.

Aberrant expression of COX-2 occurs in many types of malignancies including colon and lung cancers, and is implicated in development and progression of cancer. The molecular mechanisms associated with aberrant expression of COX-2 in lung cancer cells remain to be fully elucidated. In this study, we found that non-small cell lung cancer (NSCLC) NCI-H520 and NCI-H460 cells constitutively expressed COX-2 and produced prostaglandin E2 (PGE2) as measured by Western blotting and enzyme-linked immunosorbent assay (ELISA), respectively. Reporter assays showed that transcriptional regulation of COX-2 was blunted when either the NF-IL6 (C/EBPbeta) or nuclear factor-kappaB (NF-kappaB) binding site in the COX-2 promoter was mutated, suggesting that C/EBPbeta and NF-kappaB transcription factors have an important role in aberrant expression of COX-2 in these lung cancer cells. In addition, the eight herbal mixture PC-SPES (Lot. 5431219) caused growth arrest and apoptosis of NCI-H520 and NCI-H460 cells in association with blockade of NF-kappaB and down-regulation of C/EBPbeta, resulting in down-regulation of COX-2 and PGE2 in these cells. On the other hand, PC-SPES up-regulated the level of C/EBPbeta in these cells. Taken together, C/EBPbeta and NF-kappaB may be promising molecular targets for COX-2 inhibition in lung cancer cells. PC-SPES might be useful in the adjuvant setting for the treatment of individuals with resected NSCLC as well as other types of cancer in which COX-2 is activated.

Zanthoxyli Fructus induces growth arrest and apoptosis of LNCaP human prostate cancer cells in vitro and in vivo in association with blockade of the AKT and AR signal pathways.

Zanthoxyli Fructus belongs to the family of oranges and is used as a seasoning in Asian countries including Japan. This study found that a water extract of Zanthoxyli Fructus possessed anti-tumor activity against a wide variety of cancer cells including those from prostate (LNCaP, DU145, PC-3), breast (MCF-7, T47D, MDA-MB231), lung (NCI-H460, -H520), as well as leukemia (HL-60, NB4, Jurkat) in vitro, as measured by the trypan blue exclusion test. Importantly, Zanthoxyli Fructus slowed the proliferation of LNCaP, DU145, and MDA-MB231 cells present as xenografts in BALB/c nude mice without adverse effects. Further studies explored the molecular mechanism by which Zanthoxyli Fructus inhibited the proliferation of androgen-dependent human prostate cancer LNCaP cells because Zanthoxyli Fructus possessed the strongest anti-tumor activity against these cells. Zanthoxyli Fructus blocked androgen receptor (AR) signaling in conjunction with down-regulation of nuclear levels of AR and induced apoptosis of these cells, as measured by the reporter assay, Western blot analysis, and TUNEL assay, respectively. As expected, Zanthoxyli Fructus also decreased the level of the AR-target molecule, prostate-specific antigen in these cells. Furthermore, Zanthoxyli Fructus inhibited AKT kinase and down-regulated levels of cyclin D1 protein, as measured by the AKT kinase assay with GSK-3alpha/beta as a substrate and Western blot analysis, respectively. Taken together, Zanthoxyli Fructus might be useful as an adjunctive therapeutic agent for the treatment of individuals with a variety of cancer types.

Ganoderma lucidum causes apoptosis in leukemia, lymphoma and multiple myeloma cells.

Over many centuries, herbal remedies have treated a variety of ailments. This empiric observational approach has produced a number of leads for formulated medicines. Ganoderma lucidum extract was screened for its anti-proliferative activity using a panel of 26 human cancer cell lines. The six most sensitive hematologic cell lines were: HL-60 (ED50 26 microg/ml), U937 (63 microg/ml), K562 (50 microg/ml), Blin-1 (38 microg/ml), Nalm-6 (30 microg/ml) and RPMI8226 (40 microg/ml). Cell cycle analyses revealed a G2/M arrest, most prominently in HL-60 cells. Four hematopoietic cell lines (HL-60, Blin-1, U937, RPMI8226) were examined for apoptosis, which ranged between 21 and 92%. After exposure to G. lucidum extract, HL-60 cells became multinucleated with an increased DNA content. These results indicate that G. lucidum extract has a profound activity against leukemia, lymphoma and multiple myeloma cells and may be a novel adjunctive therapy for the treatment of hematologic malignancies.

Oridonin, a diterpenoid purified from Rabdosia rubescens, inhibits the proliferation of cells from lymphoid malignancies in association with blockade of the NF-kappa B signal pathways.

This study found that oridonin, a natural diterpenoid purified from Rabdosia rubescens, inhibited growth of multiple myeloma (MM; U266, RPMI8226), acute lymphoblastic T-cell leukemia (Jurkat), and adult T-cell leukemia (MT-1) cells with an effective dose that inhibited 50% of target cells (ED50) ranging from 0.75 to 2.7 microg/mL. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining showed that oridonin caused apoptosis of MT-1 cells in a time-dependent manner. We explored effects of oridonin on antiapoptotic Bcl-2 family members and found that it down-regulated levels of Mcl-1 and BCL-x(L), but not Bcl-2 protein, in both MT-1 and RPMI8226 cells. Further studies found that oridonin inhibited nuclear factor-kappa B (NF-kappa B) DNA-binding activity in these cells as measured by luciferase reporter gene, ELISA-based, and electrophoretic mobility shift assays. Oridonin also blocked tumor necrosis factor-alpha- and lipopolysaccharide-stimulated NF-kappa B activity in Jurkat cells as well as RAW264.7 murine macrophages. Of note, oridonin decreased survival of freshly isolated adult T-cell leukemia (three samples), acute lymphoblastic leukemia (one sample), chronic lymphocytic leukemia (one sample), non-Hodgkin's lymphoma (three samples), and MM (four samples) cells from patients in association with inhibition of NF-kappa B DNA-binding activity. On the other hand, oridonin did not affect survival of normal lymphoid cells from healthy volunteers. Taken together, oridonin might be useful as adjunctive therapy for individuals with lymphoid malignancies, including the lethal disease adult T-cell leukemia.

Retinoic acid regulates C/EBP homologous protein expression (CHOP), which negatively regulates myeloid target genes.

Retinoic acid (RA) promotes granulocytic differentiation of normal hematopoietic cells and acute promyelocytic leukemia (APL) blasts by transcriptional modulation of myeloid regulatory genes. In this study, we have identified the C/EBP homologous protein (CHOP) as a novel retinoid-responsive gene using a polymerase chain reaction (PCR)-based cDNA subtraction method. All-trans retinoic acid (ATRA) induced a biphasic expression of CHOP mRNA in the NB4 and HL60 AML cell lines. Levels of CHOP expression increased within 1 hour of exposure to ATRA. ATRA expression became nearly absent between 6 and 24 hours, and a second phase of induction occurred after 48 hours. Retinoid-dependent regulation of CHOP expression was also observed in normal human neutrophils but not in peripheral blood mononuclear cells. In addition, retinoid-dependent regulation of CHOP expression was not observed in retinoid-nonresponsive cell lines HL60R and NB4-R2. CHOP expression was regulated at the transcriptional level and was independent of new protein synthesis. CHOP heterodimerized with C/EBPepsilon and negatively regulated the myeloid-specific gene lactoferrin. Furthermore, CHOP transcriptionally inhibited C/EBPalpha- and C/EBPepsilon-dependent induction of secondary granule gene expression. RA signaling in granulocytic differentiation involves regulated expression of CHOP and C/EBPepsilon in a coordinated fashion.

Ligands for PPARgamma and RAR cause induction of growth inhibition and apoptosis in human glioblastomas.

High-grade gliomas are characterized by a rapid proliferation rate, invasiveness and angiogenesis. Our previous data indicated that the combination of ligands for peroxisome proliferator-activated receptor gamma (PPARgamma) and retinoic acid receptor (RAR) induces apoptosis of breast cancer cells in vitro and in a murine model. In this study, we have shown that 11 glioblastoma cell lines and nine fresh glioblastoma tissue samples from patients expressed high-levels of PPARgamma. In contrast, glia from nine healthy human brains expressed very low levels of PPARgamma. No mutations or polymorphisms of the PPARgamma gene were observed in these cell lines. The effect of the PPARgamma ligand Pioglitazone (PGZ) either in the absence or in the presence of a RAR ligand [all-trans retinoic acid (ATRA)] on the proliferation and apoptosis of glioblastoma cells was examined using two glioblastoma cell lines (N39 and DBTRG05MG). PGZ and/or ATRA inhibited significantly the proliferation of both cell lines. Flow cytometry analysis showed that G1 cell cycle arrest was induced by these ligands. In addition, apoptosis occurred in both cell lines treated with either PGZ or ATRA, which was associated with a downregulation of bcl-2 and an upregulation of bax proteins. An enhanced effect was observed when PGZ and ATRA were combined. Furthermore, treatment of fresh glioblastoma tissue from patients with PGZ, either alone or in combination with ATRA, induced a significant level of tumor cell apoptosis together with a downregulation of bcl-2 protein level as compared with untreated control brain tissue. Taken together, our data demonstrated that PGZ, either alone or in combination with ATRA, induced apoptosis and inhibited proliferation of glioblastoma cells, and more interestingly, induced apoptosis of fresh glioblastoma cells from patients. Therefore, we conclude that these ligands may possess adjuvant therapeutic potential for patients with glioblastoma.

PC-SPES: a potent inhibitor of nuclear factor-kappa B rescues mice from lipopolysaccharide-induced septic shock.

Septic shock is the most common cause of death in intensive care units, and no effective treatment is available at present. Lipopolysaccharide (LPS) is the primary mediator of Gram-negative sepsis by inducing the production of macrophage-derived proinflammatory cytokines, in which activation of nuclear factor-kappaB (NF-kappaB) plays an important role. PC-SPES is an eight-herb mixture active against a variety of malignancies, including prostate cancer and leukemia. In this study, we demonstrated that PC-SPES inhibited the LPS-induced NF-kappaB reporter activity in RAW264.7 macrophages. Electrophoretic mobility shift assay showed that PC-SPES inhibited the binding of NF-kappaB to specific DNA sequences; however, it did not affect either degradation of inhibitory kappaBalpha or nuclear translocation of NF-kappaB. Also, we explored the effect of PCSPES on LPS-induced mitogen-activated protein (MAP) kinase signaling; PC-SPES did not affect LPS-induced phosphorylation of MAP kinases, including c-Jun NH2-terminal kinase, p38, and extracellular signal-regulated kinase 1/2. Moreover, PC-SPES decreased the production of proinflammatory cytokines and inducible enzymes, such as tumor necrosis factor (TNF) alpha, interleukin (IL)-1beta, IL-6, cyclooxygenase-2, as well as inducible nitric-oxide synthase in RAW264.7 macrophages and peritoneal macrophages from C57BL/6 mice after the cells were stimulated by either LPS or LPS and interferon-gamma. Furthermore, PC-SPES rescued C57BL/6 mice from death caused by LPS-induced septic shock in conjunction with decreased serum levels of TNFalpha and IL-1beta. Together, PC-SPES is a potent inhibitor of NF-kappaB and might be useful for the treatment of sepsis and inflammatory diseases.

PC-SPES: Molecular mechanism to induce apoptosis and down-regulate expression of PSA in LNCaP human prostate cancer cells.

PC-SPES is an eight-herbal mixture which has activity against prostate cancer cells and can reduce the serum level of prostate specific antigen (PSA) in more than 80% of individuals with prostate cancer. We conducted this study to begin to clarify the molecular mechanism by which PC-SPES inhibited the growth of prostate cancer cells and down-regulated expression of PSA. Western blot analysis, luciferase reporter assay using a variety of promoters of the PSA gene and the isolated androgen receptor response elements (ARE), as well as electrophoretic mobility shift assay (EMSA) were employed to study the effect of PC-SPES on DHT-induced expression of PSA in LNCaP androgen-dependent human prostate cancer cells. Also, Western blot analysis and luciferase reporter assay using 12-0-tetradecanoylphorbol-13-acetate response elements were employed to study the ability of PC-SPES to activate the c-Jun NH2-terminal kinase (JNK)/c-Jun/AP-1 signal pathway in these cells. Reporter studies showed that PC-SPES inhibited DHT-induced PSA promoter/enhancer-luciferase activity via inhibition of ARE transcriptional activity. Western blot analysis showed that PC-SPES down-regulated DHT-induced expression of PSA without decreasing DHT-induced nuclear level of AR. EMSA demonstrated that PC-SPES inhibited the binding of DHT-activated AR to ARE. Moreover, we found that PC-SPES phosphorylated JNK, increased levels of phosphorylated and unphosphorylated forms of c-Jun, and enhanced AP-1 transcriptional activity in LNCaP cells. Interestingly, when LNCaP cells were stably tranfected with the dominant negative JNK binding domain (JBD) of JNK-interacting protein-1 (JIP-1), these cells no longer underwent apoptosis and growth inhibition in the presence of PC-SPES. But, PC-SPES still decreased levels of PSA in the LNCaP-JIP-1 cells. Taken together, PC-SPES inhibited binding of DHT-activated AR to AREs of PSA gene resulting in down-regulation of ARE transcriptional activity and expression of PSA, and this occurred independently of the JNK/c-Jun/AP-1 signal pathway. Also, PC-SPES activated the JNK/c-Jun/AP-1 signal pathway resulting in growth arrest and apoptosis of prostate cancer cells.

PC-SPES decreases proliferation and induces differentiation and apoptosis of human acute myeloid leukemia cells.

PC-SPES is an eight herbal mixture which has been shown to be active against prostate cancer cells in vitro as well as in patients. In this study, we discovered that it has anti-leukemia activity. HL-60, NB4, U937 and THP-1 human acute myeloid leukemia cells were cultured in the presence of various concentrations of PC-SPES (0.06-0.5 micro l/ml) for 4 days, and cell numbers were counted by Trypan blue exclusion. PC-SPES inhibited proliferation of these cells with an ED50 of 0.17, 0.09, 0.18, 0.32 micro l/ml, respectively. In clonogenic assay, PC-SPES inhibited growth of HL-60 cells (ED50, 0.043 micro l/ml). On the other hand, PC-SPES (0.1 micro l/ml) stimulated growth of normal myeloid committed stem cells (CFU-GM) by 1.4-fold of control (p=0.03). Anti-leukemia effects also occurred against freshly isolated leukemia cells from acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients. Interestingly, when PC-SPES was combined with ATRA, the antiproliferative effect was markedly enhanced. For example, PC-SPES (0.125 micro l/ml) or ATRA (10(-8) mol/l) inhibited growth of HL-60 cells after 4 days of culture, by approximately 40 and 30%, respectively; simultaneous treatment with both, suppressed growth by 80%. In addition, PC-SPES induced differentiation of HL-60 and NB4 cells, as measured by expression of CD11b and reduction of NBT. ATRA synergistically enhanced this activity. For example, either PC-SPES (0.5 micro l/ml) or ATRA (10(-8) mol/l) induced 23 and 18% of HL-60 cells, respectively to express CD11b on day 2 of culture; and when both were combined, 60% of HL-60 cells were stimulated to express CD11b antigen. Furthermore, PC-SPES (0.5 micro l/ml) produced apoptosis of HL-60 and NB4 cells, as measured by TUNEL assay, with 17% of HL-60 cells and 52% of NB4 cells becoming apoptotic on their third day of culture. Importantly, PC-SPES stimulated expression of the novel myeloid specific transcription factor C/EBPepsilon in HL-60 and NB4 cells. Taken together, PC-SPES inhibits growth and induces differentiation and apoptosis of myeloid leukemia cells, and enhances the antiproliferative and prodifferentiative effects of ATRA on these cells. PC-SPES might be useful with ATRA for treatment of patients with acute promyelocytic leukemia (APL), and it could have a role in other types of cancers including MDS.

Characterization of a myeloid tyrosine phosphatase, Lyp, and its role in the Bcr-Abl signal transduction pathway.

The Bcr-Abl protein-tyrosine kinase is implicated in the development of chronic myeloid leukemia. The potential role of protein-tyrosine phosphatase in the regulation of Bcr-Abl signaling was explored. First, expression patterns of tyrosine phosphatases in leukemic cell lines were investigated using degenerate primers for reverse transcription-PCR followed by cloning and sequencing of the cDNA. Distinct patterns of distribution of phosphatase were found in erythroid and myeloid leukemic cell lines. Whereas some phosphatases were ubiquitously expressed, others were limited to specific cell types. Surprisingly, a previously cloned "lymphocyte-specific" phosphatase, Lyp, was frequently detected in a number of myeloid cell lines as well as normal granulocytes and monocytes. Lyp was localized to the cytosol, and overexpression of Lyp caused reduction in the phosphorylation levels of multiple proteins in KCL22 chronic myeloid leukemia blast cells including Cbl, Bcr-Abl, Erk1/2, and CrkL. Co-expression of Lyp and Bcr-Abl in Cos-7 cells resulted in decreased levels of Bcr-Abl, Grb2, and Myc. Overexpression of Lyp markedly suppressed anchorage-independent clonal growth of KCL22 cells. Taken together, the data suggest that Lyp may play an antagonistic role in signaling by the Bcr-Abl fusion protein.

PC-SPES inhibits colon cancer growth in vitro and in vivo.

PC-SPES is a mixture of eight herbs with antiproliferative activity in prostate cancer cell lines and antitumor effects in animal models of prostate cancer. In addition, evidence of clinical efficacy in advanced prostate cancer has been reported. PC-SPES has also been shown to have antitumor activity against several other cancer cell lines including breast and neuroepithelial cancer, melanoma, and leukemia cell lines. Because of these findings, we investigated the effects of PC-SPES in vitro in colon cancer cell lines SW480, SW620, and DLD-1 and in vivo in the Apc(min) mouse, a murine model for intestinal carcinogenesis. For the in vitro studies, colon cancer cell lines were exposed to an ethanolic extract of PC-SPES compared with a diluent control [ethanol < or = 0.3% (v/v)]. PC-SPES resulted in a marked suppression of cell proliferation in all colon cancer cells studied. PC-SPES (3 micro l/ml) caused a 95% inhibition of cell proliferation of the DLD-1 colon cancer cell line, and similar results were observed in the SW480 and SW620 colon cancer cell lines. Cell cycle analysis demonstrated a drastic (> or =60%) accumulation of cells in the G(2)-M phase with a concomitant decrease of cells in the G(0)-G(1) phase in all colon cancer cell lines studied after treatment with PC-SPES (1.5 micro l/ml for 48 h). Western blot analysis demonstrated a decrease in protein levels of beta-tubulin in the SW620 cell line exposed to PC-SPES. Terminal deoxynucleotidyl transferase-mediated nick end labeling analysis revealed an increase in apoptotic colon cancer cells incubated with PC-SPES. For the in vivo studies, female 4-5-week-old Apc(min) mice were randomized to two groups: a PC-SPES-treated group (n = 11) received 250 mg/kg/day (0.2 ml) PC-SPES via gastrointestinal gavage; and a control group (n = 10) received 0.2 ml of the vehicle solution (1.5% carboxymethylcellulose with 0.2% Tween 20) via gastrointestinal gavage. Both groups were treated five times a week for 10 weeks. After treatment, the gastrointestinal tract was dissected for polyp scoring by two observers blinded to treatment. The Apc(min) mice given PC-SPES had a 58% reduction in tumor number and a 56% decrease in tumor load. No effect on either food intake or body weight was observed in the treated versus sham groups. The present study is the first to report the potent activity of PC-SPES against colon cancer. Both cell cycle arrest and apoptosis occurred after treatment with PC-SPES. This suggests that the components of this herbal mixture, either independently or in combination, acted in colon cancer, resulting in a drastic effect on tumor initiation and tumor progression.

Ph(+) acute lymphoblastic leukemia resistant to the tyrosine kinase inhibitor STI571 has a unique BCR-ABL gene mutation.

The tyrosine kinase inhibitor STI571 is a promising agent for the treatment of advanced Philadelphia chromosome positive (Ph(+)) acute lymphoblastic leukemia (ALL), but resistance develops rapidly in most patients after an initial response. To identify mechanisms of resistance to STI571, 30 complementary DNAs (including 9 matched samples) obtained from the bone marrow of individuals with Ph(+) ALL were analyzed by direct sequencing of a 714-base pair region of ABL encoding for the adenosine triphosphate (ATP)-binding site and the kinase activation loop. A single point mutation was found at nucleotide 1127 (GI6382056) resulting in Glu255Lys. This mutation occurred in 6 of 9 patients (67%) following their treatment with STI571 but not in the samples from patients before beginning treatment with STI571. Glu255Lys is within the motif important for forming the pocket of the ATP-binding site in ABL and it is highly conserved across species. In conclusion, Ph(+) ALL samples resistant to STI571 have a unique mutation Glu255Lys of BCR-ABL.