Table of Contents

Volume 13, Issue 13

  November 2012

  Pages 1229 - 1348

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 Issue Catalogue (MARC XML)

SPECIAL ARTICLE

Patent controversies and court cases: Cancer diagnosis, therapy and prevention

Arsenio M. Fialho and Ananda M. Chakrabarty
Pages 1229 - 1234
http://dx.doi.org/10.4161/cbt.21958
Abstract | Full Text | PDF

Patents are issued essentially by all countries on inventions that are deemed novel, non-obvious, clearly described and of significant utility or industrial application. The only exceptions to patenting an invention are abstract ideas, laws of nature and natural phenomena, although the exceptions vary depending on countries where moral, public order or human rights considerations are also taken into account. Although patent laws are updated over decades, the rapid progress of science creates situations that the patent laws on the book cannot address, leading to contentious legal issues. This is often true for life saving drugs, particularly drugs for cancers or HIV/AIDS, which are expensive and beyond the reach of poor people because of the proprietary positions of these patented drugs. Another contentious issue is the patent eligibility of human genes and mutations that are often thought of nature's contribution to human health and propagation and should be beyond the reach of patentability. In this review, we address some of these current legal issues and their implications for the development of diagnostic methods, therapeutic interventions and even prevention for cancer, a scourge of mankind.




CLINICAL STUDY

Open Access Article

K-Ras gene mutation status as a prognostic and predictive factor in patients with colorectal cancer undergoing irinotecan- or oxaliplatin-based chemotherapy

Rafał Stec, Lubomir Bodnar, Radosław Charkiewicz, Jan Korniluk, Marta Rokita, Marta Smoter, Marzena Ciechowicz, Lech Chyczewski, Jacek Nikliński, Wojciech Kozłowski and Cezary Szczylik
Pages 1235 - 1243
http://dx.doi.org/10.4161/cbt.21813
Abstract | Full Text | PDF

Background: CRC caused more than 600,000 estimated deaths in 2008. Dysregulated signaling through the RAS/RAF/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway due to mutations in K-Ras and B-Raf are common events in CRC. Methods: Incidence of mutations in codons 12 and 13 of K-Ras and exons 11 and 15 of B-Raf were analyzed in amplified PCR products from primary tumors of 273 patients with CRC, and their prognostic and predictive significance was assessed. The prognostic role of clinical and pathological factors was also examined. Results: K-Ras mutations were present in 89 patients (32.6%), of whom 76 (85.4%) had mutations in codon 12 and 10 (11.2%) had mutations in codon 13. B-Raf gene mutations were present in 17 patients (6.9%), of whom 6 (35.3%) had mutations in exon 15. Multivariate analysis revealed a predictive significance for K-Ras mutations with respect to time to progression in patients treated with irinotecan and oxaliplatin as first-line chemotherapy. There was no predictive significance for B-Raf gene mutation status in these patients. The following risk factors were found to affect overall survival (OS) rates: primary tumor location, lymph node involvement grade, carcinoembryonic antigen (CEA) level before treatment, and performance status according to WHO criteria. Conclusions: Based on the results of this study, K-Ras mutation status may be a suitable indicator of patient eligibility and a prognostic indicator for responsiveness to anti-EGFR therapy alone, or in combination with chemotherapy. Also, K-Ras mutation status may predict time to progression in patients treated with irinotecan and oxaliplatin.




RESEARCH PAPERS

Oridonin in combination with imatinib exerts synergetic anti-leukemia effect in Ph+ acute lymphoblastic leukemia cells in vitro by inhibiting activation of LYN/mTOR signaling pathway

Yong Guo, Qingqing Shan, Yuping Gong, Juan Lin, Xi Yang and Ruiqing Zhou
Pages 1244 - 1254
http://dx.doi.org/10.4161/cbt.21460
Abstract | Full Text | PDF

Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is triggered by constitutively activated BCR-ABL and SRC family tyrosine kinases.They account for the activations of multiple growth-signaling pathways, including Raf/MEK/ERK, Akt/mTOR and STAT5 pathways. The BCR-ABL tyrosine kinase inhibitor imatinib is the standard treatment for Ph+ leukemia and plays efficacious role in CML. However, imatinib has few inhibitory effects on SRC tyrosine kinase with response rate of Ph+ ALL lower and relapse more frequent and quicker compared with CML. Previous studies showed that oridonin inhibits proliferation and induces apoptosis in many tumor cells. However, the anticancer activity and mechanism of oridonin in Ph+ ALL is unknown. To investigate the anticancer activity of oridonin, we examined its role in constitutively activated Akt/mTOR, Raf/MEK/ERK, STAT5 and SRC pathway, mRNA level of bcr/abl gene, cell viability and apoptosis in Ph+ ALL SUP-B15 cells. Furthermore, we detected synergetic effect of oridonin plus imatinib. Our results showed that oridonin inhibiting activations of LYN (one of SRC family kinases) and ABL and their downstream Akt/mTOR, Raf/MEK/ERK and STAT5 pathways, downregulated Bcl-2 but upregulated Bax protein and then induced apoptosis in Ph+ ALL cells. Oridonin plus imatinib exerted synergetic effects by overcoming imatinib defect of upregulating Akt/mTOR and LYN signaling. Additionally, we examined the effect of oridonin on the signaling pathways in the primary specimens from Ph+ ALL patients. Our data showed that oridonin remarkably suppressed activations of Akt/mTOR, Raf/MEK and STAT5 pathway in these primary specimens and oridonin with imatinib exerted synergetic suppressive effects on mTOR, STAT5 and LYN signaling in one imatinib resistant patient specimen. Additional evaluation of oridonin as a potential therapeutic agent for Ph+ ALL seems warranted.

Open Access Article

A novel 7-bromoindirubin with potent anticancer activity suppresses survival of human melanoma cells associated with inhibition of STAT3 and Akt signaling

Lucy Liu, Marina Kritsanida, Prokopios Magiatis, Nicolas Gaboriaud, Yan Wang, Jun Wu, Ralf Buettner, Fan Yang, Sangkil Nam, Leandros Skaltsounis and Richard Jove
Pages 1255 - 1261
http://dx.doi.org/10.4161/cbt.21781
Abstract | Full Text | PDF

STAT3 and Akt signaling have been validated as potential molecular targets for treatment of cancers including melanoma. These small molecule inhibitors of STAT3 or Akt signaling are promising for developing anti-melanoma therapeutic agents. MLS-2438, a novel 7-bromoindirubin, a derivative of the natural product indirubin, was synthesized with a bromo-group at the 7-position on one indole ring and a hydrophilic group at the 3′-position on the other indole ring. We tested the anticancer activity of MLS-2438 and investigated its mechanism of action in human melanoma cell lines. Here, we show that MLS-2438 inhibits viability and induces apoptosis of human melanoma cells associated with inhibition of STAT3 and Akt signaling. Several pro-apoptotic Bcl-2 family proteins are involved in the MLS-2438 mediated apoptosis. MLS-2438 inhibits Src kinase activity in vitro and phosphorylation of JAK2, Src, STAT3 and Akt in cultured cancer cells. In contrast to the decreased phosphorylation levels of JAK2, Src, STAT3 and Akt, phosphorylation levels of the MAPK (Erk1/2) signaling protein were not reduced in cells treated with MLS-2438. These results demonstrate that MLS-2438, a novel natural product derivative, is a Src inhibitor and potentially regulates kinase activity of JAK2 and Akt in cancer cells. Importantly, MLS-2438 suppressed tumor growth with low toxicity in a mouse xenograft model of human melanoma. Our findings support further development of MLS-2438 as a potential small-molecule therapeutic agent that targets both STAT3 and Akt signaling in human melanoma cells.

Reporting of preclinical tumor-graft cancer therapeutic studies

Elizabeth Sugar, Adam J. Pascoe and Nilofer Azad
Pages 1262 - 1268
http://dx.doi.org/10.4161/cbt.21782
Abstract | Full Text | PDF

Purpose: Characterize the parameters of reporting tumor-graft experiments for oncologic drug development.
Experimental Design: Using Institute of Scientific Information impact factors, we identified the most-cited medical and oncology journals with tumor-graft experiments in murine models. For each article, the characteristics of the experimental design, outcome measurements, and statistical analysis were examined.
Results: We examined 145 articles describing tumor-graft experiments from October through December 2008. The articles spanned a range of disease types, animal models, treatments and delivery methods. One hundred (69%) articles were missing information needed to replicate the experiments. Outcome measurements included: tumor size (83%), biological changes (57%), and survival or cure-rate outcomes (28%). Thirty-three percent did not specify how tumor size was measured and 30% were missing the formula for evaluating volume. Only 14% utilized appropriate statistical methods. Ninety-one percent of studies were reported as positive and 7% reported with mixed positive-negative results; only 2% of studies were reported negative or inconclusive. Twenty-two articles from 2012 showed improvement in the utilization of statistical methods (35% optimal, p = 0.05) but had a similar fraction with experimental design issues (82%; p = 0.32) limiting reproducibility and 91% had positive results.
Conclusions: Tumor-graft studies are reported without a set standard, often without the methodological information necessary to reproduce the experiments. The high percentage of positive trials suggests possible publication bias. Considering the widespread use of such experiments for oncologic drug development, scientists and publishers should develop experimental and publication guidelines for such experiments to ensure continued improvements in reporting.

Development of a rat model of oral small molecule receptor tyrosine kinase inhibitor-induced diarrhea

Joanne M. Bowen, Bronwen J. Mayo, Erin Plews, Emma Bateman, Andrea M. Stringer, Frances M. Boyle, John W. Finnie and Dorothy M.K. Keefe
Pages 1269 - 1275
http://dx.doi.org/10.4161/cbt.21783
Abstract | Full Text | PDF

Orally administered small molecule receptor tyrosine kinase inhibitors (RTKIs) are increasingly common treatments for cancer, both alone and in combination with chemotherapy. However, their side effect profiles and the underlying mechanisms of such are not yet fully elucidated. Management of their most common dose limiting side effect, diarrhea, has been hampered by a lack of suitable animal models. We aimed to develop a clinically relevant rat model of RTKI-induced diarrhea that could be utilized for investigating supportive care interventions and pharmacokinetics. Albino Wistar rats were treated daily for 4 weeks with various concentrations of lapatinib to determine the optimal dose for development of diarrhea. This was then followed by an experiment with addition of paclitaxel once weekly for 4 weeks to observe effects of combination drug treatment on diarrhea. Data regarding animal tolerance to the treatment, organ weights, circulating lapatinib concentration and histopathology were collected weekly. Lapatinib caused diarrhea in rats that was dose-dependent. Diarrhea occurred without causing significant intestinal histopathology. Follow up experiments are currently underway to determine the exact pathogenesis and mechanisms of lapatinib-induced diarrhea and potential protective strategies.

CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

Hao Qian, Lijuan Xia, Peixue Ling, Samuel Waxman and Yongkui Jing
Pages 1276 - 1283
http://dx.doi.org/10.4161/cbt.21784
Abstract | Full Text | PDF

CD44 is a cell surface antigen expressed on acute myeloid leukemia cells and is used as a marker to isolate leukemia stem cells. CD44 ligation with the antibody A3D8 has been found to induce apoptosis in human acute promyelocytic leukemia (APL) cells via activation of caspase-8. The mechanism of A3D8-induced caspase-8 activation was studied in APL NB4 cells. A3D8 induces lipid raft clustering which causes Fas aggregation as determined with a confocal microscope. A3D8-induced apoptosis is abrogated by the lipid raft disrupting agent methyl-β-cyclodextrin and the caspase-8 inhibitor Z-IETD-fmk. Western blot analysis reveals that A3D8 binds to the standard form of CD44 (CD44s). HL-60 cells without detectable CD44s protein are not responsive to A3D8-induced apoptosis. SKNO-1 cells containing higher level of CD44s protein are more sensitive to A3D8-induced apoptosis than NB4 cells. These results indicate that A3D8 induces apoptosis in leukemia cells through caspase-8 activation by binding to CD44s protein and inducing lipid raft clustering.

The metabolic interactions between tumor cells and tumor-associated stroma (TAS) in prostatic cancer

Alexandra Giatromanolaki, Michael I. Koukourakis, Anastasios Koutsopoulos, Savvas Mendrinos and Efthimios Sivridis
Pages 1284 - 1289
http://dx.doi.org/10.4161/cbt.21785
Abstract | Full Text | PDF

Tumor-associated stroma (TAS) is not simply a supporting element for cancer cells, but plays an important role in tumor growth, invasion and metastasis. Changes on the level of stromal constituents, such as loss of Caveolin-1 and increased thymidine phosphorylase (TP) expression, have been associated with tumor aggressiveness. The mutual cooperation between stromal fibroblasts and cancer cells is another distinguishing feature, which has recently emerged. In this investigation, both the loss of Caveolin-1 and the increased TP expression in the prostatic TAS was associated with high Gleason score (p = 0.0002 and 0.003, respectively); the two proteins were acting both independently and synergistically. In addition, TP was significantly associated with high stromal K_i-67 (MIB1) proliferation index (p = 0.03). Analysis of the metabolic interactions between stromal and epithelial elements showed that, while prostatic cancer cells express principally (> 91%) lactate dehydrogenase-5 (LDH-5) (anaerobic metabolism), the tumor-associated fibroblasts/myofibroblasts (TAFs) express largely (67.8%) LDH-1 (aerobic metabolism)—the terms TAFs and TAS are used interchangeably. These two isoenzyme pathways act complementary; the LDH-5 pathway converts pyruvate to lactate, whereas the LDH-1 enzyme system utilizes the secreted metabolite lactate to produce pyruvate, essential for continuous energy supply to tumor cells. Monocarboxylate transporter-1 (MCT-1)—the main facilitator of lactate uptake in tumor cells, was expressed exclusively in prostate cancer cells and related directly to LDH-5 overexpression. These findings support and extend our previous studies on energy recycling between the aerobic stroma and the anaerobic cancer cells within the framework of Warburg effect.

A second target of benzamide riboside: Dihydrofolate reductase

Breton Roussel, Nadine Johnson-Farley, John E. Kerrigan, Kathleen W. Scotto, Debabrata Banerjee, Krzysztof Felczak, Krzysztof W. Pankiewicz, Murugesan Gounder, HongXia Lin, Emine Ercikan Abali and Joseph R. Bertino
Pages 1290 - 1298
http://dx.doi.org/10.4161/cbt.21786
Abstract | Full Text | PDF

Dihydrofolate reductase (DHFR) is an essential enzyme involved in de novo purine and thymidine biosynthesis. For several decades, selective inhibition of DHFR has proven to be a potent therapeutic approach in the treatment of various cancers including acute lymphoblastic leukemia, non-Hodgkin’s lymphoma, osteogenic sarcoma, carcinoma of the breast, and head and neck cancer. Therapeutic success with DHFR inhibitor methotrexate (MTX) has been compromised in the clinic, which limits the success of MTX treatment by both acquired and intrinsic resistance mechanisms. We report that benzamide riboside (BR), via anabolism to benzamide adenine dinucleotide (BAD) known to potently inhibit inosine monophosphate dehydrogenase (IMPDH), also inhibits cell growth through a mechanism involving downregulation of DHFR protein. Evidence to support this second site of action of BR includes the finding that CCRF-CEM/R human T-cell lymphoblasic leukemia cells, resistant to MTX as a consequence of gene amplification and overexpression of DHFR, are more resistant to BR than are parental cells. Studies of the mechanism by which BR lowers DHFR showed that BR, through its metabolite BAD, reduced NADP and NADPH cellular levels by inhibiting nicotinamide adenine dinucleotide kinase (NADK). As consequence of the lack of NADPH, DHFR was shown to be destabilized. We suggest that, inhibition of NADK is a new approach to downregulate DHFR and to inhibit cell growth.

Preferential induction of apoptotic cell death in melanoma cells as compared with normal keratinocytes using a non-thermal plasma torch

Shoshanna N. Zucker, Jennifer Zirnheld, Archis Bagati, Thomas M. DiSanto, Benjamin Des Soye, Joseph A. Wawrzyniak, Kasra Etemadi, Mikhail Nikiforov and Ronald Berezney
Pages 1299 - 1306
http://dx.doi.org/10.4161/cbt.21787
Abstract | Full Text | PDF

Selective induction of apoptosis in melanoma cells is optimal for therapeutic development. To achieve this goal, a non-thermal helium plasma torch was modified for use on cultured cells in a temperature-controlled environment. Melanoma cells were targeted with this torch (1) in parallel cultures with keratinocytes, (2) in co-culture with keratinocytes and (3) in a soft agar matrix. Melanoma cells displayed high sensitivity to reactive oxygen species generated by the torch and showed a 6-fold increase in cell death compared with keratinocytes. The extent of cell death was compared between melanoma cells and normal human keratinocytes in both short-term (5 min) co-culture experiments and longer assessments of apoptotic cell death (18–24 h). Following a 10 sec plasma exposure there was a 4.9-fold increase in the cell death of melanoma vs. keratinocytes as measured after 24 h at the target site of the plasma beam. When the treatment time was increased to 30 sec, a 98% cell death was reported for melanoma cells, which was 6-fold greater than the extent of cell death in keratinocytes. Our observations further indicate that this preferential cell death is largely due to apoptosis.. In addition, we report that this non-thermal plasma torch kills melanoma cells growing in soft agar, suggesting that the plasma torch is capable of inducing melanoma cell death in 3D settings. We demonstrate that the presence of gap junctions may increase the area of cell death, likely due to the “bystander effect” of passing apoptotic signals between cells. Our findings provide a basis for further development of this non-invasive plasma torch as a potential treatment for melanoma.

Open Access Article

Identification of inositol polyphosphate 4-phosphatase type II as a novel tumor resistance biomarker in human laryngeal cancer HEp-2 cells

Jae-Sung Kim, Hong Shik Yun, Hong-Duck Um, Jong Kuk Park, Kee-Ho Lee, Chang-Mo Kang, Su-Jae Lee and Sang-Gu Hwang
Pages 1307 - 1318
http://dx.doi.org/10.4161/cbt.21788
Abstract | Full Text | PDF

Although tumor resistance remains a significant impediment to successful radiotherapy, associated regulatory markers and detailed molecular mechanisms underlying this phenomenon are not well defined. In this study, we identified inositol polyphosphate 4-phosphatase type II (INPP4B) as a novel marker of radioresistance by systematically analyzing Unigene libraries of laryngeal cancer. INPP4B was highly expressed in radioresistant laryngeal cancer cells and was induced by treatment with either radiation or anticancer drugs in various types of cancer cells. Ectopic INPP4B overexpression increased radioresistance and anticancer drug resistance by suppressing apoptosis in HEp-2 cells. Conversely, INPP4B depletion with small interfering RNA resensitized HEp-2 as well as A549 and H1299 cells to radiation- and anticancer drug-induced apoptosis. Furthermore, radiation-induced INPP4B expression was blocked by inhibition of extracellular signal-regulated kinase (ERK). INPP4B depletion significantly attenuated radiation-induced increases in Akt phosphorylation, indicating an association of INPP4B-mediated radioresistance with Akt survival signaling. Taken together, our data suggest that ERK-dependent induction of INPP4B triggers the development of a tumor-resistance phenotype via Akt signaling and identify INPP4B as a potentially important target molecule for resolving the radioresistance of cancer cells.

Genetic disruption of USP9X sensitizes colorectal cancer cells to 5-fluorouracil

Dennis R. Harris, Alexandra Mims and Fred Bunz
Pages 1319 - 1324
http://dx.doi.org/10.4161/cbt.21792
Abstract | Full Text | PDF

The X-linked deubiquitinase USP9X affects the stability and activity of numerous regulatory proteins that influence cell survival. Recent studies suggest that decreased USP9X expression can confer a selective advantage onto developing cancer cells and thereby promotes disease progression. To examine the effect of USP9X on the cellular responses to anticancer therapies, we derived cancer cell lines in which the USP9X locus was disrupted by homologous recombination. The resulting USP9X-deficient cancer cells exhibited increased activation of apoptotic pathways and markedly decreased clonogenic survival in response to 5-fluorouracil, a chemotherapeutic drug that is widely used for treatment of gastrointestinal malignancies. These unexpected results suggest that cancers with low USP9X expression might be specifically sensitized to some conventional therapeutic agents.

Autophagy limits the cytotoxic effects of the AKT inhibitor AZ7328 in human bladder cancer cells

Rian J. Dickstein, Giovanni Nitti, Colin P. Dinney, Barry R. Davies, Ashish M. Kamat and David J. McConkey
Pages 1325 - 1338
http://dx.doi.org/10.4161/cbt.21793
Abstract | Full Text | PDF

Background: Mutations that activate the PI3K/AKT/mTOR pathway are relatively common in urothelial (bladder) cancers, but how these pathway mutations affect AKT dependency is not known. We characterized the relationship between AKT pathway mutational status and sensitivity to the effects of the selective AKT kinase inhibitor AZ7328 using a panel of 12 well-characterized human bladder cancer cell lines.
Methods: Sequenome DNA sequencing was performed to identify mutations in a panel of 12 urothelial cancer cell lines. Drug-induced proliferative inhibition and apoptosis were quantified using MTT assays and propidium iodide staining with FACS analyses. Protein activation via phosphorylation was measured by immunoblotting. Autophagy was measured by LC3 immunofluorescence and immunoblotting.
Results: AZ7328 inhibited proliferation and AKT substrate phosphorylation in a concentration-dependent manner but had minimal effects on apoptosis. Proliferative inhibition correlated loosely with the presence of activating PIK3CA mutations and was strengthened in combination with the mTOR inhibitor rapamycin. AZ7328 induced autophagy in some of the lines, and in the cells exposed to a combination of AZ7328 and chemical autophagy inhibitors apoptosis was induced.
Conclusions: The cytostatic effects of AZ7328 correlate with PIK3CA mutations and are greatly enhanced by dual pathway inhibition using an mTOR inhibitor. Furthermore, AZ7328 can interact with autophagy inhibitors to induce apoptosis in some cell lines. Overall, our results support the further evaluation of combinations of PI3K/AKT/mTOR pathway and autophagy inhibitors in pre-clinical in vivo models and ultimately in patients with PIK3CA mutant bladder cancers.

Dissecting the complex regulation of Mad4 in glioblastoma multiforme cells

Wensheng Yang, Xiaolu Yang, Gregory David and Jay F. Dorsey
Pages 1339 - 1348
http://dx.doi.org/10.4161/cbt.21814
Abstract | Full Text | PDF

Among proteins in the c-Myc/Max/Mad/Sin3 regulatory complex, Mad4 and Sin3B are routinely detected in human glioblastoma multiforme (GBM) cell lines. In response to gamma radiation, the expression of Sin3B and Mad4 in GBM cells was upregulated in parallel over time, suggesting that Sin3B may play a role in the regulation of Mad4 stability. In agreement with this hypothesis, exogenously expressed Sin3B significantly stabilized co-transfected Mad4 and, to a lesser extent, endogenous Mad4. In addition, siRNA silencing of Sin3B induced an increase in the expression of c-Myc and Sin3A, which contributed to increased expression of Mad4. Simultaneous silencing of Sin3B, Sin3A and c-Myc decreased Mad4 stability to a greater extent than silencing of Sin3B alone. Although Mad1 was reported to be a target of c-IAP1 E3 ligase activity for degradation, the E3 ligase activity of c-IAP1 was not required for downregulation of Mad4 expression. The association of c-IAP1 with Sin3B or Mad4 suggested that Sin3B might interfere with the binding of c-IAP1 to Mad4; however, overexpression of Sin3B did not affect the interaction between Mad4 and c-IAP1. Instead, direct binding of Sin3B to c-IAP1 may protect Mad4 from degradation by c-IAP1, leading to enhanced stability of Mad4. Exogenous expression of Sin3B also inhibited c-IAP1-mediated degradation of Mad1, TRAF2, c-IAP2 and ASK1, known targets of c-IAP1 E3 ligase activity. These results indicate that Sin3B, together with other c-Myc regulatory members, maintain the steady-state level of Mad4, in part through inhibition of c-IAP1-mediated degradation of Mad4.