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1.  Mechanisms by which IGF-I May Promote Cancer 
Cancer biology & therapy  2003;2(6):630-635.
Multiple large case-control studies in the past five years have reported positive associations between high circulating levels of the insulin-like growth factor (IGF)-I and risk for different types of cancer. Correlations certainly do not prove causation, but the reproducibility of this finding implies this is a hypothesis worth further examination through more mechanistic studies. IGF-I binds to the IGF-I receptor, a tyrosine kinase receptor that transduces signals to the nucleus and mitochondrion primarily via the mitogen-activated protein kinase (MAPK) and PI3K/Akt pathways. Examples will be provided to illustrate how IGF-I signaling may contribute to each stage of cancer progression: malignant transformation, tumor growth, local invasion and distant metastases, and resistance to treatment. In addition to direct contributions to each of these stages, IGF-I may promote cancer indirectly, through interactions with oncogenes and tumor suppressors, interactions with other hormones (especially the sex steroids in breast and prostate cancers) and interactions with the IGF binding proteins (IGFBPs). Finally, circulating IGF-I may facilitate cancer development though it likely does not cause cancer to form. Prompted by the accumulating evidence, investigations are also being pursued to modulate the IGF system as a possible means of cancer prevention or treatment.
PMCID: PMC4164051  PMID: 14688466
insulin-like growth factor (IGF); insulin-like growth factor binding proteins (IGFBPs); insulin-like growth factor receptor (IGF-1R); tyrosine kinase receptor; cancer
2.  Somatostatin and Cancer 
Cancer biology & therapy  2004;3(8):731-733.
Somatostatin is a peptide hormone that normally suppresses growth hormone (GH), thyrotropin (TSH), insulin and gut hormone release, as well as affecting multiple aspects of gastrointestinal function. It achieves these pleiotropic effects by binding somatostatin receptors (SSTR), a family of five G-protein coupled membrane receptors. Somatostatin analogs, such as octreotide, lanreotide and vapreotide, are well-established treatments for tumors that over secrete these hormones. Recently, use of somatostatin analogs for treating nonendocrine malignancies are being explored. Hu et al. found progressive reduction in SSTR3 expression when comparing normal gastric mucosa versus well differentiated versus poorly differentiated gastric adenocarcinomas; octreotide inhibited growth and induced apoptosis in vitro of those cells expressing SSTR3. Potential mechanisms by which somatostatin analogs may be useful in oncology include its endocrine actions, autocrine/paracrine effects, SSTR-mediated cell signaling and SSTR-mediated cell labeling.
PMCID: PMC4164217  PMID: 15326367
somatostatin; somatostatin receptor; growth hormone; insulin-like growth factor; insulin; gastric cancer
3.  18F and 18FDG PET imaging of osteosarcoma to non-invasively monitor in situ changes in cellular proliferation and bone differentiation upon MYC inactivation 
Cancer biology & therapy  2008;7(12):1947-1951.
Osteosarcoma is one of the most common pediatric cancers. Accurate imaging of osteosarcoma is important for proper clinical staging of the disease and monitoring of the tumor’s response to therapy. The MYC oncogene has been commonly implicated in the pathogenesis of human osteosarcoma. Previously, we have described a conditional transgenic mouse model of MYC-induced osteosarcoma. These tumors are highly invasive and are frequently associated with pulmonary metastases. In our model, upon MYC inactivation osteosarcomas lose their neoplastic properties, undergo proliferative arrest, and differentiate into mature bone. We reasoned that we could use our model system to develop noninvasive imaging modalities to interrogate the consequences of MYC inactivation on tumor cell biology in situ. We performed positron emission tomography (PET) combining the use of both 18F-fluorodeoxyglucose (18FDG) and 18F-flouride (18F) to detect metabolic activity and bone mineralization/remodeling. We found that upon MYC inactivation, tumors exhibited a slight reduction in uptake of 18FDG and a significant increase in the uptake of 18F along with associated histological changes. Thus, these cells have apparently lost their neoplastic properties based upon both examination of their histology and biologic activity. However, these tumors continue to accumulate 18FDG at levels significantly elevated compared to normal bone. Therefore, PET can be used to distinguish normal bone cells from tumors that have undergone differentiation upon oncogene inactivation. In addition, we found that 18F is a highly sensitive tracer for detection of pulmonary metastasis. Collectively, we conclude that combined modality PET/CT imaging incorporating both 18FDG and 18F is a highly sensitive means to non-invasively measure osteosarcoma growth and the therapeutic response, as well as to detect tumor cells that have undergone differentiation upon oncogene inactivation.
PMCID: PMC4158945  PMID: 18981708
MYC; osteosarcoma; PET; 18F; 18FDG; trangenic mouse model; metastasis
4.  Anti-oxidant treatment enhances anti-tumor cytotoxicity of (−)-gossypol 
Cancer biology & therapy  2008;7(5):767-776.
We showed that tumor cells with wild-type p53 and high levels of Bcl-xL are cisplatin resistant but are induced to undergo apoptosis by (−)-gossypol, making this a promising agent for overcoming cisplatin resistance. However, some cells in a population with this phenotype are not killed and continue to survive. Conversely, tumor cells with low Bcl-xL expression and either wild type or mutant p53 are relatively cisplatin sensitive and do not exhibit such high levels of apoptosis. However, these do undergo progressive loss of viability after (−)-gossypol that may not be tumor specific. We sought to elucidate the basis for these observations using cDNA microarray analysis of (−)-gossypol treated cisplatin sensitive and resistant cells. Genes in the reactive oxygen species (ROS) pathway were highly up-regulated in response to (−)-gossypol. The up-regulation was of much greater magnitude in cisplatin sensitive than resistant cells. Staining with an oxidation reporter dye confirmed differential induction of ROS in tumor cells with low Bcl-xL. As (−)-gossypol is known to undergo oxidative metabolism in vivo, ROS generation may be responsible for both off-target cytotoxicity and inactivation of the drug. In agreement with this hypothesis, oxidation of (−)-gossypol by pre-treatment with hydrogen peroxide eliminated its activity. Combined treatment with the antioxidant N-acetyl-cysteine (NAC) to block ROS increased (−)-gossypol-induced cytotoxicity to tumor but not normal cells. Furthermore, NAC increased the induction of apoptosis as measured by the sub G1 population, in both cisplatin sensitive and resistant cells. We postulate that concurrent treatment with antioxidant to block ROS prevents oxidative inactivation of (−)-gossypol and limits off-target toxicity allowing more potent (−)-gossypol-induced anti tumor activity.
PMCID: PMC4157391  PMID: 18376141
Cisplatin resistance; (−)-gossypol; Bcl-xL; reactive oxygen species; head and neck squamous cell carcinoma; apoptosis
5.  BMP Suppresses PTEN Expression via RAS/ERK Signaling 
Cancer biology & therapy  2007;6(8):1313-1317.
Bone morphogenetic protein (BMP), a member of the transforming growth factor β family, classically utilizes the SMAD signaling pathway for its growth suppressive effects, and loss of this signaling cascade may accelerate cell growth. In the colon cancer predisposition syndrome Juvenile Polyposis, as well as in the late progression stages of nonsyndromic colorectal cancers, SMAD4 function is typically abrogated. Here, we utilized the SMAD4-null SW480 colon cancer cell line to examine BMPs effect on a potential target gene, PTEN, and how its expression might be regulated. Initial treatment of the SMAD4-null cells with BMP resulted in mild growth suppression, but with prolonged exposure to BMP, the cells become growth stimulatory, which coincided with observed decreases in transcription and translation of PTEN, and with corresponding increases in phospho-AKT protein levels. BMP-induced PTEN suppression was mediated via the RAS/ERK pathway, as pharmacologic inhibition of RAS/ERK, or interference with protein function in the cytosol by DN-RAS prevented BMP-induced growth promotion and changes in PTEN levels, as did treatment with noggin, a BMP ligand inhibitor. Thus, BMP downregulates PTEN via RAS/ERK in a SMAD4-null environment that contributes to cell growth, and constitutes a SMAD4-independent but BMP-responsive signaling pathway.
PMCID: PMC4154563  PMID: 18059158
bone morphogenetic protein; PTEN; RAS; ERK; TGFβ; colon cancer
6.  Pre-Clinical Evaluation of 1-Nitroacridine Derived Chemotherapeutic Agent that has Preferential Cytotoxic Activity Towards Prostate Cancer 
Cancer biology & therapy  2007;6(10):1632-1637.
Chemotherapy in prostate cancer (CaP) even as an adjunct has not been a success. In this communication, we report the pre-clinical efficacy of a nitroacridine derivative, C-1748 (9[2′-hydroxyethylamino]-4-methyl-1-nitroacridine) in CaP cell culture and human xenograft animal models. C-1748, a DNA intercalating agent has been derived from its precursor C-857 that was a potent anti-cancer drug, but failed clinical development “methyl” group imparted novel properties, the most interesting of which is the difference in the IC50 values between LnCaP (22.5 nM), a CaP cell line and HL-60, a leukemia cell line (>100 nM). Using γH2AX as an intervention marker of DNA double strand breaks, we concluded that C-1748 is more efficacious in CaP cells than in HL-60 cells. In hormone dependent cells, the androgen receptor (AR) was identified as an additional target of C-1748. In xenograft studies, administration of C-1748 intra-peritoneally inhibited tumor growth by 80–90% with minimal toxicity. These studies identify C-1748 as a novel acridine drug that has a high therapeutic index and low cytotoxicity on myelocytic cells with potential for clinical development.
PMCID: PMC4134887  PMID: 17921700
chemotherapy; xenografts; 1-nitroacridine derivative; systemic treatment; DNA double strand breaks; γH2AX
7.  Preclinical and Clinical Studies with the Multi-Kinase Inhibitor CEP-701 as Treatment for Prostate Cancer Demonstrate the Inadequacy of PSA Response as a Primary Endpoint 
Cancer biology & therapy  2007;6(9):1360-1367.
CEP-701 is a potent inhibitor of trk receptors that causes cell death in prostate cancer (PC) models. CEP-701 binds to serum proteins and a preprostatectomy study was performed to assess prostate tissue penetration and clinical response to CEP-701.
Growth assays and Western blot analyses were performed to evaluate CEP-701 kinase inhibition. In a preprostatectomy study, patients received CEP-701 for five days prior to prostatectomy and prostate tissue analyzed for CEP-701 levels. A phase II dose escalation study was performed in patients with hormone refractory PC with rising PSA and no metastases. Endpoints included PSA response and safety.
CEP-701 binds to serum proteins limiting tissue penetration. An oral dose of 40 mg bid of CEP-701 for five days produced levels of 219 ± 38 nM in prostate at time of prostatectomy. No patients in the Phase II study met the primary response criteria of >50% PSA decline. Seven/9 patients had increase in PSA slope on CEP-701 compared to PSA slope prestudy. Five/9 patients had a decrease in PSA levels after stopping CEP-701. Laboratory studies showed increased PSA production by CEP-701 growth arrested human PC cells in vitro and in vivo.
Evaluation of PSA response is an inadequate indicator of response in CEP-701 treated PC patients. Therefore, the effectiveness of CEP-701 as treatment for prostate cancer has not been adequately tested. Based on a strong preclinical rationale, further clinical studies with CEP-701 using alternative endpoints are indicated.
PMCID: PMC4124640  PMID: 17786033
prostate cancer; PSA; kinase; peceptor; CEP-701
8.  Non-Invasive Fluorescence Imaging of Cell Death in Fresh Human Colon Epithelia Treated with 5-Fluorouracil, CPT-11 and/or TRAIL 
Cancer biology & therapy  2005;4(9):937-942.
Apoptosis is instrumental in several physiological/pathophysiological processes and is a frequently used end-point in the development of anti-neoplastic compounds. Despite ample data on several colon cancer cell lines, little is known about the susceptibility of human colon to apoptosis following treatment with established chemotherapeutics. By treating fresh human colonic explants with 5-Fluorouracil (200 μg/ml), CPT-11 (100 μg/ml) and/or TRAIL (100 ng/ml) we readily detected a signal in situ using FITC-VAD-FMK at different time points, whereas labeling of colonic explants with EGFP-conjugated Annexin V proved less specific. Although TRAIL treatment alone appeared to cause little apoptosis in human colonic epithelia versus the control, we observed a greater number of cells undergoing apoptosis when a combination of CPT-11 and TRAIL was used as compared to either agent alone. This is the initial demonstration of TRAIL-induced apoptosis with or without a chemotherapeutic agent in fresh primary human colon epithelia explants. Thus, human colonic explants may provide a valuable reference point when candidate therapeutic compounds triggering apoptosis in colon cancer cell lines, xenografts or mouse models are developed. The results support the feasibility of developing non-invasive optical imaging strategies to detect apoptosis through direct visualization of injury to human colonic epithelia in vivo.
PMCID: PMC4121850  PMID: 16251801
non-invasive imaging; fluorescence imaging; apoptosis; cancer therapy; drug toxicity; colon epithelia; FLICA; TRAIL
9.  Apogossypolone, a nonpeptidic small molecule inhibitor targeting Bcl-2 family proteins, effectively inhibits growth of diffuse large cell lymphoma cells in vitro and in vivo 
Cancer biology & therapy  2008;7(9):1418-1426.
Apogossypolone (ApoG2) is a semi-synthesized derivative of gossypol. The principal objective of this study was to compare stability and toxicity between ApoG2 and gossypol, and to evaluate anti-lymphoma activity of ApoG2 in vitro and in vivo. ApoG2 shows better stability when compared with a racemic gossypol and can be better tolerated by mice compared to gossypol. ApoG2 showed significant inhibition of cell proliferation of WSU-DLCL2 and primary cells obtained from lymphoma patients, whereas it displayed no toxicity on normal peripheral blood lymphocytes. For a treatment of 72 h, the IC50 of ApoG2 was determined to be 350 nM against WSU-DLCL2 cells. Treatment with ApoG2 at 600 mg/kg resulted in significant growth inhibition of WSU-DLCL2 xenografts. When combined with CHOP, ApoG2 displayed even more complete inhibition of tumor growth. ApoG2 binds to purified recombinant Bcl-2, Mcl-1 and Bcl-XL proteins with high affinity and is shown to block the formation of heterodimers between Bcl-XL and Bim. For a treatment of 72 h, ApoG2 induced a maximum of 32% of apoptotic cell death. Western blot experiments showed that treatment with ApoG2 led to cleavage of caspase-3, caspase-9 and PARP. Moreover, pretreatment of DLCL2 cells with caspase-3, -9 and broad spectrum caspase inhibitors significantly blocked growth inhibition induced by ApoG2. In conclusion, ApoG2 effectively inhibits growth of DLCL2 cells at least partly by inducing apoptosis. It is an attractive small molecule inhibitor of the Bcl-2 family proteins to be developed further for the treatment of diffuse large cell lymphoma.
PMCID: PMC4106024  PMID: 18769131
small molecule inhibitors; Bcl-2 family of protein; diffuse large cell lymphoma; apoptosis; chemotherapy; animal model; toxicity
10.  Allele-specific expression in the germline of patients with familial pancreatic cancer 
Cancer biology & therapy  2007;7(1):135-144.
Physiologic allele-specific expression (ASE) in germline tissues occurs during random X-chromosome inactivation1 and in genomic imprinting,2 wherein the two alleles of a gene in a heterozygous individual are not expressed equally. Recent studies have confirmed the existence of ASE in apparently non-imprinted autosomal genes;3–14 however, the extent of ASE in the human genome is unknown. We explored ASE in lymphoblastoid cell lines of 145 individuals using an oligonucleotide array based assay. ASE of autosomal genes was found to be a very common phenomenon in ∼20% of heterozygotes at 78% of SNPs at 84% of the genes examined. Comparison of 100 affected individuals from familial pancreatic cancer kindreds and 45 controls revealed three types of changes in the germline: (a) loss of ASE, (b) gain of ASE, and, (c) rare instances of “extreme” (near monoallelic) ASE. The latter changes identified heterozygous deleterious mutations in a subset of these genes. Consequently, an ASE assay efficiently identifies candidate disease genes with altered germline expression properties as compared to controls, and provides insights into mechanisms that confer an inherited disease risk for pancreatic cancer.
PMCID: PMC4104667  PMID: 18059179
allele-specific; familial; pancreatic; mutation; microarray; cancer; regulation
11.  Dasatinib inhibits both osteoclast activation and prostate cancer PC-3 cell-induced osteoclast formation 
Cancer biology & therapy  2009;8(22):2153-2159.
Therapies to target prostate cancer bone metastases have only limited effects. New treatments are focused on the interaction between cancer cells, bone marrow cells and the bone matrix. Osteoclasts play an important role in the development of bone tumors caused by prostate cancer. Since Src kinase has been shown to be necessary for osteoclast function, we hypothesized that dasatinib, a Src family kinase inhibitor, would reduce osteoclast activity and prostate cancer (PC-3) cell-induced osteoclast formation.
Dasatinib inhibited RANKL-induced osteoclast differentiation of bone marrow-derived monocytes with an EC50 of 7.5 nM. PC-3 cells, a human prostate cancer cell line, were able to differentiate RAW 264.7 cells, a murine monocytic cell line, into osteoclasts and dasatinib inhibited this differentiation. In addition, conditioned medium from PC-3 cell cultures was able to differentiate RAW 264.7 cells into osteoclasts and this too, was inhibited by dasatinib. Even the lowest concentration of dasatinib, 1.25 nmol, inhibited osteoclast differentiation by 29%. Moreover, dasatinib inhibited osteoclast activity by 58% as measured by collagen 1 release.
Experimental design
We performed in vitro experiments utilizing the Src family kinase inhibitor dasatinib to target osteoclast activation as a means of inhibiting prostate cancer bone metastases.
Dasatinib inhibits osteoclast differentiation of mouse primary bone marrow-derived monocytes and PC-3 cell-induced osteoclast differentiation. Dasatinib also inhibits osteoclast degradation activity. Inhibiting osteoclast differentiation and activity may be an effective targeted therapy in patients with prostate cancer bone metastases.
PMCID: PMC4073296  PMID: 19855158
osteoclast; Src; prostate cancer; dasatinib; PC-3
12.  Delocalization of γ-tubulin due to increased solubility in human breast cancer cell lines 
Cancer biology & therapy  2010;9(1):66-76.
The centrosome is the major organelle responsible for the nucleation and organization of microtubules into arrays. Recent studies demonstrate that microtubules can nucleate outside the centrosome. The molecular mechanisms controlling acentrosomal microtubule nucleation are currently poorly defined, and the function of this type of microtubule regulation in tumor cell biology is particularly unclear. Since microtubule nucleation is initiated by the gamma-tubulin protein, we examined the regulation of gamma-tubulin in a panel of human breast tumor cell lines, ranging from non-tumorigenic to highly aggressive. We have identified a more dispersive subcellular localization of gamma-tubulin in aggressive breast cancer cell lines, while gamma-tubulin localization remains largely centrosomal in non-invasive cell lines. Delocalization of gamma-tubulin occurs independently from changes in protein expression and is therefore regulated at the post-translational level. Subcellular fractionation revealed that tumor cell lines show an aberrantly increased release of gamma-tubulin into a soluble cytoplasmic fraction, with the most dramatic changes observed in tumor cell lines of greater metastatic potential. Extraction of soluble gamma-tubulin revealed acentrosomal incorporation of gamma-tubulin in cytoplasmic microtubules and along cell junctions. Moreover, acentrosomal delocalization of gamma-tubulin yielded resistance to colchicine-mediated microtubule collapse. These findings support a model where the solubility of gamma-tubulin can be altered through post-translational modification and provides a new mechanism for microtubule dysregulation in breast cancer. Gamma-tubulin which is delocalized from the centrosome can still clearly be incorporated into filaments, and defines a novel mechanism for tumor cells to develop resistance to microtubule-targeted chemotherapies.
PMCID: PMC4073665  PMID: 20009567
γ-tubulin; centrosomes; microtubule nucleation; breast cancer; solubility
13.  Mitogen activated protein kinase phosphatases and cancer 
Cancer biology & therapy  2010;9(5):337-340.
Deregulation of cell signaling is a vital part of cancer development. The mitogen activated protein kinase (MAPK) family is involved in regulating both cell growth and cell death. This family of kinases is negatively regulated by mitogen activated protein kinase phosphatases (MKPs). MKPs are dual specificity phosphatases that target threonine and tyrosine residues that appear in a TXY motif. There are eleven members of the MKP family. Expression of MKPs has been shown to be altered in many different types of cancer. Most of what is known centers on MKP-1, MKP-2 and MKP-3. This review will focus on their role in cancer development and progression.
PMCID: PMC4063278  PMID: 20139719
MKP-1; MKP-2; MKP-3; dual specificity phosphatases; MAPK signaling; cancer
14.  The Effects of the Histone Deacetylase Inhibitor Romidepsin (FK228) Are Enhanced by Aspirin (ASA) in COX-1 Positive Ovarian Cancer Cells through Augmentation of p21 
Cancer biology & therapy  2010;9(11):928-935.
Histone deacetylase (HDAC) inhibitors have shown preclinical efficacy in solid tumors, including ovarian cancers. Our group has published that the HDAC inhibitor, romidepsin (FK228) suppresses ovarian cancer cell growth at nanomolar concentrations in vitro. HDAC inhibitors appear to be even more effective when used in combination with other anti-tumor agents. However, it remains unclear which anti-tumor agents are best suited for combination therapy. A recent report suggested that aspirin (acetylsalicylic acid, ASA) is synergistic with HDAC inhibitors in ovarian cancer cells. ASA is a relatively selective inhibitor of cyclooxygenase-1 (COX-1) and has anti-proliferative effects in ovarian cancer cells. The goal of this study was to investigate the impact of ASA on the activity of the HDAC inhibitor, FK228 in COX-1 positive (OVCAR-3) and COX-1 negative (SKOV-3) human ovarian cancer cell lines. The growth inhibitory effects of FK228 were enhanced by ASA in COX-1 positive ovarian cancer cells. In contrast, ASA had no influence on the results of FK228 treatment in COX-1 negative ovarian cancer cells. Upregulation of the cell cycle control protein p21 was induced robustly by FK228 in both cell lines. In the COX-1 positive cells, p21 expression was augmented by the addition of ASA to FK228 treatment. Furthermore, COX-1 siRNA attenuated the effects of combined ASA and FK228 on the levels of p21 expression and the amount of growth inhibition. The additional increase in p21 by ASA in FK228-treated cells was not observed at the promoter or transcriptional levels. However, a significant delay in p21 protein degradation in the presence of ASA and FK228 in COX-1 positive cells was associated with inhibition of proteasome activity. Our study provides a potential rationale for combining ASA with HDAC inhibitors in a subset of ovarian cancers.
PMCID: PMC4047717  PMID: 20404564
Ovarian cancer; romidepsin; aspirin; p21; HDAC inhibitors; COX inhibitors; cell cycle control
15.  Modulating the tumor microenvironment to increase radiation responsiveness 
Cancer biology & therapy  2009;8(21):1994-2001.
Radiosensitivity can be influenced both by factors intrinsic and extrinsic to the cancer cell. One of the factors in the tumor microenvironment (TME) extrinsic to the cancer cell that can affect radiosensitivity is oxygenation. Severely hypoxic cells require a 2–3 fold higher dose of radiation to achieve the same level of cell killing as do well-oxygenated cells. Other elements in the microenvironment that may influence tumor radiosensitivity are the response of stromal cells to radiation and the expression of factors such as vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1 (HIF-1). There are currently several classes of agents that may increase tumor radiosensitivity by modulating the TME. Pre-clinical evidence indicates that inhibition of VEGF may increase local control after radiation. Several mechanisms have been postulated to explain this including radiosensitization of tumor endothelial cells, prevention of the establishment of new vasculature post-radiation, and increased oxygenation secondary to vascular normalization. Agents targeting HIF-1 also increase local control after radiation in pre-clinical models. This may occur via indirect inhibition of VEGF, which is a downstream target of HIF-1, or by VEGF-independent means. When combined with radiation, the EGFR inhibitor cetuximab improves local control and survival in patients with head and neck cancer. Pre-clinical data indicate that EGFR inhibitors can increase the intrinsic radiosensitivity of cancer cells. They can also improve tumor blood flow and oxygenation, which may increase extrinsic radiosensitivity. One of the pathways downstream of EGFR that may contribute to this effect is the PI3K/Akt pathway. Agents that directly inhibit this pathway improve blood flow and increase tumor oxygenation in pre-clinical models. The challenge remains to obtain clinical data from patients showing that modulation of the TME is an important mechanism by which biological agents can radiosensitize tumors and then to utilize this information to optimize therapy.
PMCID: PMC3965173  PMID: 19823031
radiation; radiosensitization; vascular normalization; EGFR; VEGF; HIF; PI3 kinase; Akt; tumor microenvironment
16.  Docosahexaenoic acid induces dose dependent cell death in an early undifferentiated subtype of acute myeloid leukemia cell line 
Cancer biology & therapy  2009;8(4):331-337.
Acute myeloid leukemia (AML) is the most frequently diagnosed adulthood leukemia, yet current therapies offer a cure rate of less than 30%. This may be due in part to the fact that the leukemia-initiating cells in AML reside within the rare and highly primitive CD34+CD38- hematopoietic stem/progenitor cell (HSC) population that are often resistant to chemotherapy. Docosahexanoic acid (DHA), a major component of fish oil, has previously been shown to inhibit the induction and progression of breast, prostate and colon cancer, and increase the therapeutic effects of numerous chemotherapeutics, often by enhancing apoptosis. In the present studies, we investigated DHA's effect on the primitive and undifferentiated AML cell line KG1a, to explore the potential of this fatty acid to serve as adjuvant therapy for AML. Treatment of KG1a cells with DHA for 96 hours did not lead to maturation or cell cycle modification when compared to an untreated KG1a control (n = 4). However, DHA treatment of KG1a cells resulted in a progressive loss of viability, DNA fragmentation, and an increase in Annexin V expression, demonstrating DHA-induced apoptosis (n = 4). Moreover, expression of the pro-apoptotic protein Bax was increased, with resultant skewing in the Bax/bcl-2 ratio, providing a mechanistic explanation for the observed DHA-induced increase in apoptosis. Since we also show that DHA does not have a detrimental effect on normal hematopoiesis our results suggest that DHA could potentially serve as an well-tolerated adjuvant in the treatment of AML patients.
PMCID: PMC3954154  PMID: 19197149
docosahexaenoic acid; DHA; KG1a; acute myeloid leukemia; apoptosis; Bax; Bcl2
17.  Suppression of Proliferation of Two Independent NF1 Malignant Peripheral Nerve Sheath Tumor Cell Lines by the pan-ErbB Inhibitor CI-1033 
Cancer biology & therapy  2008;7(12):1938-1946.
Neurofibromatosis Type 1 (NF1) is characterized by the abnormal proliferation of neuroectodermal tissues and the development of certain malignancies, particularly neurofibromas, which may progress into malignant peripheral nerve sheath tumors (MPNSTs). Effective pharmacological therapy for the treatment of NF1 tumors is currently unavailable, and the prognosis for patients with MPNSTs is poor. Loss of neurofibromin correlates with increased expression of the epidermal growth factor receptor (EGFR) and ErbB2 tyrosine kinases, and these kinases have been shown to promote NF1 tumor-associated pathologies in vivo. We show here that while NF1 MPNST cells have higher EGFR expression levels and are more sensitive to EGF when compared to a non-NF1 MPNST cell line, the ability of the EGFR inhibitor gefitinib to selectively inhibit NF1 MPNST cell proliferation is marginal. We also show that NF1 MPNST proliferation correlates with activated ErbB2, and can be suppressed by nanomolar concentrations of the pan-ErbB inhibitor CI-1033 (canertinib). Consequently, targeting both EGFR and ErbB2 may prove an effective strategy for suppressing NF1 MPNST tumor growth in vivo.
PMCID: PMC3923431  PMID: 18927496
EGF receptor; ErbB2; NF1; tyrosine kinase inhibitor; MPNST
18.  The silent estrogen receptor 
Cancer biology & therapy  2009;8(6):485-496.
PMCID: PMC3901993  PMID: 19411863
breast cancer; epigenetic silencing; hormone resistance; ER reactivation; growth factors
19.  Inhibition of MCL-1 enhances lapatinib toxicity and overcomes lapatinib resistance via BAK-dependent autophagy 
Cancer biology & therapy  2009;8(21):2084-2096.
Prior studies demonstrated that resistance to the ERBB1/2 inhibitor Lapatinib in HCT116 cells was mediated by increased MCL-1 expression. We examined whether inhibition of BCL-2 family function could restore Lapatinib toxicity in Lapatinib adapted tumor cells and enhance Lapatinib toxicity in naive cells. The BCL-2 family antagonist Obatoclax (GX15-070), that inhibits BCL-2/BCL-Xl/MCL-1 function, enhanced Lapatinib toxicity in parental HCT116 and Lapatinib adapted HCT116 cells. In breast cancer lines, regardless of elevated ERBB1/2 expression, GX15-070 enhanced Lapatinib toxicity within 3–12 h.The promotion of Lapatinib toxicity neither correlated with cleavage of caspase 3 nor was blocked by inhibition caspases; and was not associated with changes in the activities of ERK1/2, JNK1/2 or p38 MAPK but with reduced AKT, mTOR and S6K1 phosphorylation. The promotion of Lapatinib toxicity by GX15-070 correlated with increased cytosolic levels of apoptosis inducing factor (AIF) and expression of ATG8 (LC3), and the formation of large vesicles that intensely stained for a transfected LC3-GFP construct. Knockdown of the autophagy regulatory proteins ATG5 or Beclin1 suppressed the induction of LC3-GFP vesicularization and significantly reduced cell killing, whereas knock down of MCL-1 and BCL-Xl enhanced the induction of LC3-GFP vesicularization and significantly enhanced cell killing. Knockdown of Beclin1 and AIF abolished cell killing. Collectively, our data demonstrate that Obatoclax mediated inhibition of MCL-1 rapidly enhances Lapatinib toxicity in tumor cells via a toxic form of autophagy and via AIF release from the mitochondrion.
PMCID: PMC3887451  PMID: 19823038
lapatinib; obatoclax; autophagy; cell death; resistance
20.  Diversity of DNA damage response of astrocytes and glioblastoma cell lines with various p53 status to treatment with etoposide and temozolomide 
Cancer biology & therapy  2009;8(5):452-457.
Phpsphorylation of histone H2AX is a sensitive marker of DNA damage, particularly of DNA double strand breaks. Using multiparameter cytometry we explored effects of etoposide and temozolomide (TMZ) on three glioblastoma cell lines with different p53 status (A172, T98G, YKG-1) and on normal human astrocytes (NHA) correlating the drug-induced phosphorylated H2AX (γH2AX) with cell cycle phase and induction of apoptosis. Etoposide induced γH2AX in all phases of the cell cycle in all three glioblastoma lines and led to an arrest of T98G and YKG-1 cells in S and G2/M. NHA cells were arrested in G1 with no evidence of γH2AX induction. A172 responded by rise in γH2AX throughout all phases of the cycle, arrest at the late S- to G2/M-phase, and appearance of senescence features: induction of p53, p21WAF1/CIP1, p16INK4A and β-galactosidase, accompanied by morphological changes typical of senescence. T98G cells showed the presence of γH2AX in S phase with no evidence of cell cycle arrest. A modest degree of arrest in G1 was seen in YKG-1 cells with no rise in γH2AX. While frequency of apoptotic cells in all four TMZ-treated cell cultures was relatively low it is conceivable that the cells with extensive DNA damage were reproductively dead. The data show that neither the status of p53 (wild-type vs. mutated, or inhibited by pifithrin-α) nor the expression of O6-methylguanine-DNA methyltransferase significantly affected the cell response to TMZ. Because of diversity in response to TMZ between individual glioblastoma lines our data suggest that with better understanding of the mechanisms, the treatment may have to be customized to individual patients.
PMCID: PMC3855308  PMID: 19305157
glioblastoma; temozolomide; etoposide; DNA double strand break; DNA damage; senescence; cell cycle
21.  Mirk/Dyrk1B, a novel therapeutic target, mediates cell survival in non-small cell lung cancer cells 
Cancer biology & therapy  2009;8(17):1671-1679.
Minibrain-related kinase (Mirk) is a member of the dual specificity tyrosine-phosphorylation-regulated kinase (Dyrk)/minibrain family of dual-specificity protein kinases and is identical to Dyrk1B. Mirk/Dyrk1B is a serine/threonine kinase that has been found to be upregulated in solid tumors and mediates cell survival in colon cancer, pancreatic ductal adenocarcinoma and rhabdomyosarcomas. There is little known about Mirk in lung cancer. In the present study, we showed that Mirk protein was widely overexpressed in 13 of 19 NSCLC cell lines. Mirk immunoprecipitation coupled with anti-phosphotyrosine western blotting confirmed tyrosine phosphorylation of Mirk in NSCLC cells. Mirk knockdown by small interfering RNA induced cell apoptosis concomitant with upregulation of Bak, a Bcl-2 family member, and downregulation of signal transducers and activators of transcription 3 (STAT3) tyrosine phosphorylation. Mirk knockdown led to decreased cell colony formation in vitro as well as delayed tumor growth in an orthotopic mouse model and sensitized cells to cisplatin-induced apoptosis. Using automated quantitative determination of the Mirk protein in tumor specimens of patients with early-stage lung cancer, overexpression of Mirk was found in nearly 90% of tumor specimens in both the cytoplasm and nucleus. These results suggest that Mirk is overexpressed in lung cancer, acts as a survival factor in lung cancer cells and may be a novel therapeutic target.
PMCID: PMC3839311  PMID: 19633423
Mirk/Dyrk1B; STAT3; Bcl-2; siRNA; apoptosis; lung cancer
22.  Cancer stem cells and hepatocellular carcinoma 
Cancer biology & therapy  2009;8(18):1691-1698.
Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide, with a median survival of 6–16 m. Factors responsible for the poor prognosis include late onset diagnosis, underlying cirrhosis and resistance to chemotherapy; 40% of HCCs are clonal and therefore potentially arise from progenitor/stem cells. New insights are provided from several signaling pathways, such as STAT3, NOTCH, hedgehog and transforming growth factor-β (TGFβ), which are involved in stem cell renewal, differentiation, survival, and are commonly deregulated in HCC. Control of stem cell proliferation by the TGFβ, Notch, Wnt and Hedgehog pathways to suppress hepatocellular cancer and to form the endoderm suggest a dual role for this pathway in tumor suppression as well as progression of differentiation from a stem or progenitor stage. This review provides a rationale for detecting and analyzing tumor stem cells as one of the most effective ways to treat cancers such as hepatocellular cancer.
PMCID: PMC3821558  PMID: 19901516
TGF; 2-SPECTRIN; stem/progenitor cells; hepatocellular cancer; signal transduction
23.  Mcl1 downregulation sensitizes neuroblastoma to cytotoxic chemotherapy and small molecule Bcl2-family antagonists 
Cancer biology & therapy  2009;8(16):1587-1595.
Neuroblastoma (NB) is a common, highly lethal pediatric cancer, with treatment failures largely attributable to the emergence of chemoresistance. The pro-survival Bcl2 homology (BH) proteins critically regulate apoptosis, and may represent important therapeutic targets for restoring drug sensitivity in NB. We used a human NB tumor tissue microarray to survey the expression of pro-survival BH proteins Mcl1 and Bcl2, and correlated expression to clinical prognostic factors and survival. Primary NB tumors heterogeneously expressed Mcl1 or Bcl2, with high expression correlating to high-risk phenotype. Co-expression is infrequent (11%), but correlates to reduced survival. Using RNA interference, we investigated the functional relevance of Mcl1 and Bcl2 in high-risk NB cell lines (SK-N-AS, IMR-5, NLF). Mcl1 knockdown induced apoptosis in all NB cell lines, while Bcl2 knockdown inhibited only NLF, suggesting functional heterogeneity Finally, we determined the relevance of Mcl1 in resistance to conventional chemotherapy (etoposide, doxorubicin) and small molecule Bcl2-family antagonists (ABT-737 and AT-101). Mcl1 silencing augmented sensitivity to chemotherapeutics 2- to 300-fold, while Bcl2 silencing did not, even in Bcl2-sensitive NLF cells. Resistance to ABT-737, which targets Bcl2/-w/-x, was overcome by Mcl1 knockdown. AT-101, which also neutralizes Mcl1, had single-agent cytotoxicity, further augmented by Mcl1 knockdown. In conclusion, Mcl1 appears a predominant pro-survival protein contributing to chemoresistance in NB, and Mcl1 inactivation may represent a novel therapeutic strategy. Optimization of compounds with higher Mcl1 affinity, or combination with additional Mcl1 antagonists, may enhance the clinical utility of this approach.
PMCID: PMC3770183  PMID: 19556859
embryonal tumors; Bcl2 family; oncogene; experimental therapeutics; Bcl2 antagonists; neuroblastoma; Mcl1
24.  A combined, rational approach towards inhibition of the MEK-ERK and mTOR pathways in pancreatic ductal adenocarcinoma 
Cancer biology & therapy  2009;8(20):1902-1903.
PMCID: PMC3720128  PMID: 19783905
pancreatic cancer; transgenic mouse; MEK; Kras; mTOR
25.  The role of autophagy as a mechanism of cytotoxicity by the clinically used agent MDA-7/IL-24 
Cancer biology & therapy  2010;9(7):537-538.
PMCID: PMC3682463  PMID: 20234182
autophagy; apoptosis; MDA-7/IL-24; glioblastoma multiforme; PERK signaling

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