bluuebunny/crossref_metadata_2025 huggingface dataset:
Aberrant hemichannel properties of Cx26 mutations causing skin disease and deafness
Authors: Gerido Dwan A. , DeRosa Adam M. , Richard Gabriele , White Thomas W.
Published: April 2007

Mutations in the human GJB2 gene, which encodes connexin26 (Cx26), underlie various forms of hereditary deafness and skin disease. While it has proven difficult to discern the exact pathological mechanisms that cause these disorders, studies have shown that the loss or abnormal function of Cx26 protein has a profound effect on tissue homeostasis. Here, we used the Xenopus oocyte expression system to examine the functional characteristics of a Cx26 mutation (G45E) that results in keratitis-ichthyosis-deafness syndrome (KIDS) with a fatal outcome. Our data showed that oocytes were able to express both wild-type Cx26 and its G45E variant, each of which formed hemichannels and gap junction channels. However, Cx26-G45E hemichannels displayed significantly greater whole cell currents than wild-type Cx26, leading to cell lysis and death. This severe phenotype could be rescued in the presence of elevated Ca2+ levels in the extracellular milieu. Cx26-G45E could also form intercellular channels with a similar efficiency as wild-type Cx26, however, with increased voltage sensitive gating. We also compared Cx26-G45E with a previously described Cx26 mutant, A40V, which has an overlapping human phenotype. We found that both dominant Cx26 mutants elicited similar functional consequences and that cells coexpressing mutant and wild-type connexins predominantly displayed mutant-like behavior. These data suggest that mutant hemichannels may act on cellular homeostasis in a manner that can be detrimental to the tissues in which they are expressed.
Effects of corticospinal tract stimulation on renal sympathetic nerve activity in rats with intact and chronically lesioned spinal cords
Authors: Pan Baohan , Zahner Matthew R. , Kulikowicz Ewa , Schramm Lawrence P.
Published: April 2007

Sympathetic preganglionic neurons and interneurons are closely apposed (presumably synapsed upon) by corticospinal tract (CST) axons. Sprouting of the thoracic CST rostral to lumbar spinal cord injuries (SCI) substantially increases the incidence of these appositions. To test our hypothesis that these additional synapses would increase CST control of sympathetic activity after SCI, we measured the effects of electrical stimulation of the CST on renal sympathetic nerve activity (RSNA) and arterial pressure (AP) in α-chloralose-anesthetized rats with either chronically intact or chronically lesioned spinal cords. Stimuli were delivered to the CST at intensities between 25–150 μA and frequencies between 25 and 75 Hz. Stimulation of the CST at the midcervical level decreased RSNA and AP. These decreases were not mediated by direct projections of the CST to the thoracic spinal cord because we could still elicit them by midcervical stimulation after acute lesions of the CST at caudal cervical levels. In contrast, caudal thoracic CST stimulation increased RSNA and AP. Neither the responses to cervical nor thoracic stimulation were affected by chronic lumbar SCI. These data show that the CST mediates decreases in RSNA via a cervical spinal system but excites spinal sympathetic neurons at caudal thoracic levels. Because chronic lumber spinal cord injury affected responses evoked from neither the cervical nor thoracic CST, we conclude that lesion-induced or regeneration-induced formation of new synapses between the CST and sympathetic neurons may not affect cardiovascular regulation.
Temporal-spatial expression of ANG-(1-7) and angiotensin-converting enzyme 2 in the kidney of normal and hypertensive pregnant rats
Authors: Joyner J. , Neves L. A. A. , Granger J. P. , Alexander B. T. , Merrill D. C. , Chappell M. C. , Ferrario C. M. , Davis W. P. , Brosnihan K. B.
Published: April 2007

We recently demonstrated that renin-angiotensin system (RAS) overactivity during late gestation in rats is associated with increased kidney and urine levels of ANG-(1-7) and enhanced kidney immunostaining of ANG-(1-7) and angiotensin-converting enzyme 2 (ACE2). To understand the temporal-spatial changes in normal and hypertensive pregnancies, the renal distribution of ANG-(1-7) and ACE2 in association with kidney angiotensin peptides and ACE2 activity was examined in virgin, normal pregnant (NP; gestational days 5, 15, and 19) and reduced uterine perfusion pressure (RUPP at day 19) pregnant Sprague-Dawley rats. ANG-(1-7) and ACE2 immunocytochemical staining increased 1.8- and 1.9-fold and 1.7- and 1.8-fold, respectively, at days 15 and 19 of NP, compared with virgin rats. ANG-(1-7) and ANG II concentrations were increased in the kidney at 19 days of gestation. ACE2 activity measured using a fluorescent substrate was increased 1.9- and 1.9-fold in the cortex and 1.9- and 1.8-fold in the medulla at days 15 and 19 of NP. In the RUPP animals, ANG-(1-7) immunostaining and concentration were significantly decreased compared with 19-day NP rats. ACE2 activity was unchanged in the cortex and medulla of RUPP rats. In conclusion, during NP, the concurrent changes of ACE2 and ANG-(1-7) suggest that ACE2 plays an important role in regulating the renal levels of ANG-(1-7) at mid to late gestation. However, the decrease in renal ANG-(1-7) content in the absence of a concomitant decrease in ACE2 implicates the participation of other ANG-(1-7) forming or degrading enzymes during hypertensive pregnancy.
Limiting sarcolemmal Na+entry during resuscitation from ventricular fibrillation prevents excess mitochondrial Ca2+accumulation and attenuates myocardial injury
Authors: Wang Sufen , Radhakrishnan Jeejabai , Ayoub Iyad M. , Kolarova Julieta D. , Taglieri Domenico M. , Gazmuri Raúl J.
Published: April 2007

Background: intracellular Na+accumulation during ischemia and reperfusion leads to cytosolic Ca2+overload through reverse-mode operation of the sarcolemmal Na+-Ca2+exchanger. Cytosolic Ca2+accumulation promotes mitochondrial Ca2+(Ca2+m) overload, leading to mitochondrial injury. We investigated whether limiting sarcolemmal Na+entry during resuscitation from ventricular fibrillation (VF) attenuates Ca2+moverload and lessens myocardial dysfunction in a rat model of VF and closed-chest resuscitation. Methods: hearts were harvested from 10 groups of 6 rats each representing baseline, 15 min of untreated VF, 15 min of VF with chest compression given for the last 5 min (VF/CC), and 60 min postresuscitation (PR). VF/CC and PR included four groups each randomized to receive before starting chest compression the new NHE-1 inhibitor AVE4454B (1.0 mg/kg), the Na+channel blocker lidocaine (5.0 mg/kg), their combination, or vehicle control. The left ventricle was processed for intracellular Na+and Ca2+mmeasurements. Results: limiting sarcolemmal Na+entry attenuated cytosolic Na+increase during VF/CC and the PR phase and prevented Ca2+moverload yielding levels that corresponded to 77% and 71% of control hearts at VF/CC and PR, without differences among specific Na+-limiting interventions. Limiting sarcolemmal Na+entry attenuated reductions in left ventricular compliance during VF and prompted higher mean aortic pressure (110 ± 7 vs. 95 ± 11 mmHg, P < 0.001) and higher cardiac work index (159 ± 34 vs. 126 ± 29 g·m·min−1·kg−1, P < 0.05) with lesser increases in circulating cardiac troponin I at 60 min PR. Conclusions: Na+-limiting interventions prevented excess Ca2+maccumulation induced by ischemia and reperfusion and ameliorated myocardial injury and dysfunction.
Voltage-Sensitive Conductances of Bushy Cells of the Mammalian Ventral Cochlear Nucleus
Authors: Cao Xiao-Jie , Shatadal Shalini , Oertel Donata
Published: April 2007

Bushy cells in the ventral cochlear nucleus convey firing of auditory nerve fibers to neurons in the superior olivary complex that compare the timing and intensity of sounds at the two ears and enable animals to localize sound sources in the horizontal plane. Three voltage-sensitive conductances allow bushy cells to convey acoustic information with submillisecond temporal precision. All bushy cells have a low-voltage-activated, α-dendrotoxin (α-DTX)-sensitive K+conductance ( gKL) that was activated by depolarization past −70 mV, was half-activated at −39.0 ± 1.7 (SE) mV, and inactivated ∼60% over 5 s. Maximal gKLvaried between 40 and 150 nS (mean: 80.8 ± 16.7 nS). An α-DTX-insensitive, tetraethylammonium (TEA)-sensitive, K+conductance ( gKH) was activated at voltages positive to −40 mV, was half-activated at −18.1 ± 3.8 mV, and inactivated by 90% over 5 s. Maximal gKHvaried between 35 and 80 nS (mean: 58.2 ± 6.5 nS). A ZD7288-sensitive, mixed cation conductance ( gh) was activated by hyperpolarization greater than −60 mV and half-activated at −83.1 ± 1.1 mV. Maximum ghranged between 14.5 and 56.6 nS (mean: 30.0 ± 5.5 nS). 8-Br-cAMP shifted the voltage sensitivity of ghpositively. Changes in temperature stably altered the steady-state magnitude of Ih. Both gKLand gKHcontribute to repolarizing action potentials and to sharpening synaptic potentials. Those cells with the largest ghand the largest gKLfired least at the onset of a depolarization, required the fastest depolarizations to fire, and tended to be located nearest the nerve root.
HVC Neural Sleep Activity Increases With Development and Parallels Nightly Changes in Song Behavior
Authors: Crandall Shane R. , Adam Murtaza , Kinnischtzke Amanda K. , Nick Teresa A.
Published: April 2007

Sleep abnormalities are coexpressed with human communication disorders. Recent data from the birdsong system, the best model for human speech, indicate that sleep has a critical role in vocal learning. To understand the neural mechanisms that underlie behavioral changes during sleep, we recorded sleep activity in the song control area HVC longitudinally during song development in zebra finches. We focused on the sensorimotor phase of song learning, when the finch shapes his song behavior toward a learned tutor song model. Direct comparison of sleep activity in adults and juveniles revealed that the juvenile HVC has a lower spike rate and longer silent periods than the adult. Within individual finches, sleep silent periods decreased and spike rate increased with age. We next systematically compared neural sleep activity and song behavior. We now report that spike rate during sleep was significantly correlated with overnight changes in song behavior. Collectively, these data indicate that sleep activity in the vocal control area HVC increases with age and may affect song behavior.
Electrophysiological Properties and Subunit Composition of GABAAReceptors in Patients With Gelastic Seizures and Hypothalamic Hamartoma
Authors: Wu Jie , Chang Yongchang , Li Guohui , Xue Fenqin , DeChon Jamie , Ellsworth Kevin , Liu Qiang , Yang Kechun , Bahadroani Nasim , Zheng Chao , Zhang Jianliang , Rekate Harold , Rho Jong M. , Kerrigan John F.
Published: April 2007

Abnormalities in GABAAreceptor structure and/or function have been associated with various forms of epilepsy in both humans and animals. Whether this is true for patients with gelastic seizures and hypothalamic hamartoma (HH) is unknown. In this study, we characterized the pharmacological properties and native subunit composition of GABAAreceptors on acutely dissociated single neurons from surgically resected HH tissues using patch-clamp, immunocytochemical, and RT-PCR techniques. We found that 1) GABA induced an inward current ( IGABA) at a holding potential of −60 mV; 2) IGABAwas mimicked by the GABAAreceptor agonist muscimol and blocked by the GABAAreceptor antagonist bicuculline, suggesting that IGABAwas mediated principally through the GABAAreceptor; 3) the EC50and Hill coefficient derived from the IGABAconcentration-response curve were 6.8 μM and 1.9, respectively; 4) the current-voltage curve was linear at a reversal potential close to zero; and 5) IGABAexhibited low sensitivity to zinc and diazepam but higher sensitivity to pentobarbital and pregnanolone. Additionally, using Xenopus oocytes microtransplanted with normal human hypothalamic tissue, we confirmed that the functional properties of GABAAreceptors were similar to those seen in small isolated HH neurons. Finally, the expression profile of GABAAreceptor subunits obtained from normal control human hypothalamic tissue was identical to that from surgically resected human HH tissue. Taken together, our data indicate that GABAAreceptors on small HH neurons exhibit normal pharmacosensitivity and subunit composition. These findings bear relevance to a broader understanding of inhibitory neurotransmission in human HH tissue.
Bilateral Vestibular Loss in Cats Leads to Active Destabilization of Balance During Pitch and Roll Rotations of the Support Surface
Authors: Macpherson Jane M. , Everaert Dirk G. , Stapley Paul J. , Ting Lena H.
Published: April 2007

Although the balance difficulties accompanying vestibular loss are well known, the underlying cause remains unclear. We examined the role of vestibular inputs in the automatic postural response (APR) to pitch and roll rotations of the support surface in freely standing cats before and in the first week after bilateral labyrinthectomy. Support surface rotations accelerate the body center of mass toward the downhill side. The normal APR consists of inhibition in the extensors of the uphill limbs and excitation in the downhill limbs to decelerate the body and maintain the alignment of the limbs with respect to earth-vertical. After vestibular lesion, cats were unstable during rotation perturbations and actively pushed themselves downhill rather than uphill, using a postural response that was opposite to that seen in the control trials. The extensors of the uphill rather than downhill limbs were activated, whereas those of the downhill limbs were inhibited rather than being excited. We propose that vestibular inputs provide an important reference to earth-vertical, which is critical to computing the appropriate postural response during active orientation to the vertical. In the absence of this vestibular information, subjects orient to the support surface using proprioceptive inputs, which drives the body downhill resulting in instability and falling. This is consistent with current models of sensory integration for computation of body posture and orientation.
Spatial Frequency-Specific Contrast Adaptation Originates in the Primary Visual Cortex
Authors: Duong Thang , Freeman Ralph D.
Published: April 2007

Adaptation to a high-contrast grating stimulus causes reduced sensitivity to subsequent presentation of a visual stimulus with similar spatial characteristics. This behavioral finding has been attributed by neurophysiological studies to processes within the visual cortex. However, some evidence indicates that contrast adaptation phenomena are also found in early visual pathways. Adaptation effects have been reported in retina and lateral geniculation nucleus (LGN). It is possible that these early pathways could be the physiological origin of the cortical adaptation effect. To study this, we recorded from single neurons in the cat's LGN. We find that contrast adaptation in the LGN, unlike that in the visual cortex, is not spatial frequency specific, i.e., adaptation effects apply to a broad range of spatial frequencies. In addition, aside from the amplitude attenuation, the shape of spatial frequency tuning curves of LGN cells is not affected by contrast adaptation. Again, these findings are unlike those found for cells in the visual cortex. Together, these results demonstrate that pattern specific contrast adaptation is a cortical process.
TIP30 Mutant Derived from Hepatocellular Carcinoma Specimens Promotes Growth of HepG2 Cells through Up-Regulation of N-cadherin
Authors: Jiang Chao , Pecha Jill , Hoshino Isamu , Ankrapp David , Xiao Hua
Published: April 2007

TIP30 is a tumor suppressor whose expression is altered in human liver, prostate, lung, colon, and breast cancers. Mice lacking TIP30 spontaneously developed hepatocellular carcinomas (HCC) and other tumors at a higher incidence than wild-type mice. Somatic missense mutations in the TIP30 gene were identified in human HCC tissue specimens, which resulted in instability or abnormal cellular distribution of TIP30 protein in cells. Here, we show that TIP30 mutants are able to promote cell growth and invasion and inhibit cisplatin-induced apoptosis in the HCC cell line HepG2 negative for endogenous TIP30. Moreover, one of the TIP30 mutants can dramatically accelerate tumor formation in immunodeficient mice. Analysis of gene expression in HepG2 cells, ectopically expressing either wild-type TIP30 or mutant TIP30, by Affymetrix GeneChip array, real-time quantitative PCR, and Western blotting assays reveals that TIP30 mutants can alter expression of genes involved in the regulation of tumorigenesis. This includes up-regulation of expression of N-cadherin and c-MYC and down-regulation of expression of p53 and E-cadherin. N-cadherin knockdown with small interfering RNA in HepG2 cells expressing mutant TIP30 resulted in a profound reduction in cell viability. Taken together, our data indicate that somatic mutations in the TIP30 gene may abolish its native tumor-suppressor activity and gain oncogenic activities partially through up-regulation of N-cadherin, thereby potentiating the pathogenesis of HCC in patients. [Cancer Res 2007;67(8):1–9]
Monocyte Chemotactic Protein-1 Mediates Prostate Cancer–Induced Bone Resorption
Authors: Lu Yi , Cai Zhong , Xiao Guozhi , Keller Evan T. , Mizokami Atsushi , Yao Zhi , Roodman G. David , Zhang Jian
Published: April 2007

Prostate cancer preferentially metastasizes to bone, resulting in high mortality. Strategies to inhibit prostate cancer metastasis include targeting both tumor-induced osteoblastic lesions and underlying osteoclastic activities. We and others have previously shown that blocking receptor activator of nuclear factor-κB ligand (RANKL) partially blocks tumor establishment and progression in bone in murine models. However, levels of RANKL in the cell lines used in these studies were very low, suggesting that soluble factors other than RANKL may mediate the cancer-induced osteoclast activity. To identify these factors, a human cytokine antibody array was used to measure cytokine expression in conditioned medium collected from primary prostate epithelial cells (PrEC), prostate cancer LNCaP and its derivative C4-2B, and PC3 cells. All prostate cancer cells produced high amounts of monocyte chemotactic protein-1 (MCP-1) compared with PrEC cells. Furthermore, levels of interleukin (IL)-6, IL-8, GROα, ENA-78, and CXCL-16 were higher in PC3 than LNCaP. These results were confirmed by ELISA. Finally, human bone marrow mononuclear cells (HBMC) were cultured with PC3 conditioned medium. Although both recombinant human MCP-1 and IL-8 directly stimulated HBMC differentiation into osteoclast-like cells, IL-8, but not MCP-1, induced bone resorption on dentin slices with 21 days of culture in the absence of RANKL. However, the conditioned medium–induced bone resorption was inhibited by MCP-1 neutralizing antibody and was further synergistically inhibited with IL-8 antibody, indicating that MCP-1, in addition to IL-8, mediates tumor-induced osteoclastogenesis and bone resorption. MCP-1 may promote preosteoclast cell fusion, forming multinucleated tartrate-resistant acid phosphatase–positive osteoclast-like cells. This study may provide novel therapeutic targets for treatment of prostate cancer skeletal metastasis. [Cancer Res 2007;67(8):3646–53]
Modulation of Angiogenic Phenotype Alters Tumorigenicity in Rat Ovarian Epithelial Cells
Authors: Schumacher Jennifer J. , Dings Ruud P.M. , Cosin Jonathan , Subramanian Indira V. , Auersperg Nelly , Ramakrishnan Sundaram
Published: April 2007

Vascular endothelial growth factor (VEGF) expression correlates with microvessel density, stage, malignant ascites, metastasis, and survival in ovarian cancer. By transducing VEGF165 into a nontumorigenic rat ovarian surface epithelial cell line (ROSE199), we investigated the direct effect of an angiogenic phenotype on tumor development. The neu oncogene, which is overexpressed in >30% of ovarian cancers, was used in comparison. Neu-transfected ROSE199 cells showed phenotypic characteristics of transformation in vitro with an abundance of focus-forming units in monolayer cultures and anchorage-independent growth in soft agar. In contrast, VEGF-secreting ROSE199 cells (VR) retained normal morphology and in vitro growth characteristics (e.g., proliferation rate) compared with parental ROSE199 cells. Interestingly, injection of VR cells into athymic mice formed malignant ascites in 100% of the animals when injected into the peritoneum and developed vascularized tumors in 85% of the mice when injected s.c. Furthermore, blocking VEGF-mediated signaling by the Flk-1/KDR receptor kinase inhibitor SU5416 significantly inhibited the growth of VR tumors. To validate that the proangiogenic switch is responsible for tumor development, the angiogenic phenotype was balanced by the inducible coexpression of endostatin under the control of Tet-activated promoter. Coexpression of endostatin along with VEGF reversed the tumorigenic phenotype of VR cells. These studies show that alterations in the angiogenic characteristics of ovarian surface epithelium may play an important role in the etiology of ovarian cancer, and that inhibition of angiogenesis can be effective in the treatment of epithelial ovarian cancer. [Cancer Res 2007;67(8):3683–90]
Sanguinarine-Dependent Induction of Apoptosis in Primary Effusion Lymphoma Cells
Authors: Hussain Azhar R. , Al-Jomah Naif A. , Siraj Abdul K. , Manogaran Pulicat , Al-Hussein Khalid , Abubaker Jehad , Platanias Leonidas C. , Al-Kuraya Khawla S. , Uddin Shahab
Published: April 2007

Primary effusion lymphoma (PEL) is an incurable, aggressive B-cell malignancy that develops rapid resistance to conventional chemotherapy. In efforts to identify novel approaches to block proliferation of PEL cells, we found that sanguinarine, a natural compound isolated from the root plant Sanguinaria canadendid, inhibits cell proliferation and induces apoptosis in a dose-dependent manner in several PEL cell lines. Our data show that sanguinarine treatment of PEL cells results in up-regulation of death receptor 5 (DR5) expression via generation of reactive oxygen species (ROS) and causes activation of caspase-8 and truncation of Bid (tBid). Subsequently, tBid translocates to the mitochondria causing conformational changes in Bax, leading to loss of mitochondrial membrane potential and release of cytochrome c to the cytosol. Sanguinarine-induced release of cytochrome c results in activation of caspase-9 and caspase-3 and poly(ADP-ribose) polymerase (PARP) cleavage, leading to induction of caspase-dependent apoptosis. In addition, we show that pretreatment of PEL cells with carbobenzoxy-Val-Ala-Asp-fluoromethylketone, a universal inhibitor of caspases, abrogates caspase and PARP activation and prevents cell death induced by sanguinarine. Moreover, treatment of PEL cells with sanguinarine down-regulates expression of inhibitor of apoptosis proteins (IAP). Finally, N-acetylcysteine, an inhibitor of ROS, inhibits sanguinarine-induced generation of ROS, up-regulation of DR5, Bax conformational changes, activation of caspase-3, and down-regulation of IAPs. Taken together, our findings suggest that sanguinarine is a potent inducer of apoptosis of PEL cells via up-regulation of DR5 and raise the possibility that this agent may be of value in the development of novel therapeutic approaches for the treatment of PEL. [Cancer Res 2007;67(8):3888–97]
An Imaging Biomarker of Early Treatment Response in Prostate Cancer that Has Metastasized to the Bone
Authors: Lee Kuei C. , Sud Sudha , Meyer Charles R. , Moffat Bradford A. , Chenevert Thomas L. , Rehemtulla Alnawaz , Pienta Kenneth J. , Ross Brian D.
Published: April 2007

Prostate cancer ranks as the most common lethal malignancy diagnosed and the second leading cause of cancer mortality in American men. Although high response rates are achieved using androgen blockade as first-line therapy, most men progress toward hormone-refractory prostate cancer. Systemic chemotherapies have been shown to improve clinical outcome in hormone refractory prostate cancer patients; however, they are not curative. Due to the high incidence of bone involvement in hormone-refractory prostate cancer, assessment of treatment response in metastatic prostate cancer to the bone remains a major clinical need. In this current study, we investigated the feasibility of using the functional diffusion map (fDM) as an imaging biomarker for assessing early treatment response in a preclinical model of metastatic prostate cancer. The fDM biomarker requires a pretreatment and midtreatment magnetic resonance imaging diffusion map, which is used to quantify spatially distinct therapeutic-induced changes in the Brownian motion (or diffusion) of water within tumor tissue. Because water within tumor cells is in a restricted environment relative to extracellular water, loss of cell membrane integrity and cellular density during therapy will be detected by fDM as an increase in diffusion. Regions of significantly increased diffusion values were detected early using fDM in docetaxel-treated versus untreated metastatic prostate bone tumors at 7 days post treatment initiation (P < 0.05), indicating loss of tumor cell viability. Validation of fDM results was accomplished by histologic analysis of excised tissue. Results from this study show the capability of fDM as a biomarker for detection of bone cancer treatment efficacy, thus warranting clinical evaluation. [Cancer Res 2007;67(8):3524–8]
Identification of Cancer Stem Cell–Like Side Population Cells in Human Nasopharyngeal Carcinoma Cell Line
Authors: Wang Jing , Guo Li-Ping , Chen Li-Zhen , Zeng Yi-Xin , Lu Shih Hsin
Published: April 2007

Side population (SP) cells have been isolated from several solid tumors. They lack distinct molecular markers for cancer stem cells (CSC) and increasing evidence suggests that they may play an important role in tumorigenesis and cancer therapy. However, there are no reports about the existence and function of SP cells in nasopharyngeal carcinoma (NPC) cells thus far. In this study, we scanned SP cells from five NPC cell lines and investigated stem cell characteristics, such as proliferation, self-renewal, and differentiation, using SP cells from the widely-used CNE-2 NPC cell line. We observed a strong tumorigenesis ability of SP cells following in vivo transplantation into nonobese diabetic/severe combined immunodeficient mice. Immunofluorescence revealed that cytokine 19 was highly expressed on SP cells. SP cells were found to be more resistant to chemotherapy and radiotherapy and this was related to the ATP-binding cassette half transporter member 2 of G family protein and Smoothened protein expression, respectively. Our results not only showed that SP cells in human NPC cell line CNE-2 had stem cell characteristics in vitro but also showed that they had a strong ability to form tumors in vivo. Importantly, we found the cell marker, cytokine 19, may serve as a potential molecular marker for further characterization of CSC. Taken together, our data shed light on tumorigenesis and therapeutic-resistant mechanisms, which are helpful for developing novel targets for effective clinical treatment of NPC. [Cancer Res 2007;67(8):3716–24]
GRP78/BiP Inhibits Endoplasmic Reticulum BIK and Protects Human Breast Cancer Cells against Estrogen Starvation–Induced Apoptosis
Authors: Fu Yong , Li Jianze , Lee Amy S.
Published: April 2007

The recent development of hormonal therapy that blocks estrogen synthesis represents a major advance in the treatment of estrogen receptor–positive breast cancer. However, cancer cells often acquire adaptations resulting in resistance. A recent report reveals that estrogen starvation–induced apoptosis of breast cancer cells requires BIK, an apoptotic BH3-only protein located primarily at the endoplasmic reticulum (ER). Searching for novel partners that interact with BIK at the ER, we discovered that BIK selectively forms complex with the glucose-regulated protein GRP78/BiP, a major ER chaperone with prosurvival properties naturally induced in the tumor microenvironment. GRP78 overexpression decreases apoptosis of 293T cells induced by ER-targeted BIK. For estrogen-dependent MCF-7/BUS breast cancer cells, overexpression of GRP78 inhibits estrogen starvation–induced BAX activation, mitochondrial permeability transition, and consequent apoptosis. Further, knockdown of endogenous GRP78 by small interfering RNA (siRNA) sensitizes MCF-7/BUS cells to estrogen starvation–induced apoptosis. This effect was substantially reduced when the expression of BIK was also reduced by siRNA. Our results provide the first evidence that GRP78 confers resistance to estrogen starvation–induced apoptosis in human breast cancer cells via a novel mechanism mediated by BIK. These results further suggest that GRP78 expression level in the tumor cells may serve as a prognostic marker for responsiveness to hormonal therapy based on estrogen starvation and that combination therapy targeting GRP78 may enhance efficacy and reduce resistance. [Cancer Res 2007;67(8):3734–40]
Elucidating the Function of Secreted Maspin: Inhibiting Cathepsin D–Mediated Matrix Degradation
Authors: Khalkhali-Ellis Zhila , Hendrix Mary J.C.
Published: April 2007

Cellular interaction with the extracellular milieu plays a significant role in normal biological and pathologic processes. Excessive degradation of basement membrane matrix by proteolytic enzymes is a hallmark of tumor invasion and metastasis, and aspartyl proteinase cathepsin D is implicated as a major contributor to this process. Maspin, a non-inhibitory serpin, plays an important role in mammary gland development and remodeling. Expression of Maspin is decreased in primary tumors and lost in metastatic lesions. Maspin is mostly cytoplasmic and is partially secreted; however, the fate and function of secreted Maspin has remained mostly unexplored. We hypothesized that secreted Maspin is incorporated into the matrix deposited by normal mammary epithelial cells and thus could play a critical role in cathepsin D–mediated matrix degradation and remodeling of mammary tissue. In the absence of Maspin, as is the case with most cancer cells, matrix degradation proceeds unrestricted, thus facilitating the progression to metastasis. To test this, we employed an in vitro model where gels containing both types I and IV collagen were preconditioned with normal mammary epithelial cells to allow the incorporation of secreted Maspin. This conditioned matrix was used to examine cathepsin D–mediated collagen degradation by human breast cancer cell lines. Our results indicate that secretion of Maspin and its deposition into the extracellular milieu play an important role in matrix degradation. In this capacity, Maspin could potentially regulate mammary tissue remodeling occurring under normal and pathologic conditions. In addition, these findings could have a potential effect on future therapeutic intervention strategies for breast cancer. [Cancer Res 2007;67(8):3535–9]
GRP78 Induction in Cancer: Therapeutic and Prognostic Implications
Authors: Lee Amy S.
Published: April 2007

Cancer cells adapt to chronic stress in the tumor microenvironment by inducing the expression of GRP78/BiP, a major endoplasmic reticulum chaperone with Ca2+-binding and antiapoptotic properties. GRP78 promotes tumor proliferation, survival, metastasis, and resistance to a wide variety of therapies. Thus, GRP78 expression may serve as a biomarker for tumor behavior and treatment response. Combination therapy suppressing GRP78 expression may represent a novel approach toward eradication of residual tumors. Furthermore, the recent discovery of GRP78 on the cell surface of cancer cells but not in normal tissues suggests that targeted therapy against cancer via surface GRP78 may be feasible. [Cancer Res 2007;67(8):3496–9]
The randomized discontinuation trial: a phase II design to assess growth-inhibitory agents
Authors: Stadler Walter M.
Published: April 2007

An increasing number of putative anticancer targets and drugs have been identified with many of these expected to be growth inhibitory. Clinical development of these agents in the phase II setting is challenging because tumor shrinkages, or at least tumor shrinkages that meet the standard definitions of objective response, are not expected. Time to progression end points are however problematic because expected times in the absence of therapy (the null hypothesis) cannot be predicted accurately, thus requiring trials to enroll a concurrent control group. Another problem is that the patient population that will benefit from a new drug remains poorly defined in early-phase development. The randomized discontinuation trial design addresses both of these issues. All patients are initially treated with the drug; patients with an objective response continue therapy; patients who do not progress or experience excess toxicity within a prespecified “run-in” period are then randomized to continuing or discontinuing therapy in a double-blind, placebo controlled manner. Despite certain limitations that need to be recognized, the ability of this design to “select” a cohort most likely to benefit and to rigorously evaluate the disease-stabilizing activity of an investigational agent provides multiple advantages. [Mol Cancer Ther 2007;6(4):1180–5]
Molecular mechanisms of the chemopreventive effect on hepatocellular carcinoma development in Mdr2 knockout mice
Authors: Katzenellenbogen Mark , Mizrahi Lina , Pappo Orit , Klopstock Naama , Olam Devorah , Barash Hila , Domany Eytan , Galun Eithan , Goldenberg Daniel
Published: April 2007

Dietary antioxidants and selenium compounds were shown to have a therapeutic effect against hepatocellular carcinoma in several mouse models. We tested the effects of tannic acid and selenomethionine on hepatocellular carcinoma development in Mdr2 knockout (Mdr2-KO) mice. Mdr2-KO and age-matched Mdr2 heterozygous control mice were fed with tannic acid or selenomethionine during the first 3 months of life. Then, several mice from each group were sacrificed, and liver tissue samples were removed for analysis. The remaining mice were fed a regular diet until the age of 16 months, at which time the number and size of liver tumors were determined. Liver tissue samples of 3-month-old mice were subjected to gene expression profiling analysis using cDNA macroarrays containing probes for 240 genes that regulate responses to oxidative stress and inflammation or lipid metabolism. Both tannic acid and selenomethionine had partial chemopreventive effect on development of hepatocellular carcinoma in Mdr2-KO mice: they reduced the incidence of large tumor nodules (diameter >1 cm) at age 16 months. Both agents inhibited gene expression and reversed up-regulation of many genes that control inflammation or response to oxidative stress in Mdr2-KO livers at age 3 months. This inhibitory effect on gene expression correlated with the ability of agents to reduce incidence of large tumors: selenomethionine was more active than tannic acid in both aspects. Understanding the molecular mechanism of chemoprevention effect could improve our therapeutic modalities while using these agents. [Mol Cancer Ther 2007;6(4):1283–91]
Liposomal curcumin with and without oxaliplatin: effects on cell growth, apoptosis, and angiogenesis in colorectal cancer
Authors: Li Lan , Ahmed Bilal , Mehta Kapil , Kurzrock Razelle
Published: April 2007

The role of curcumin (diferuloylmethane), a proapoptotic compound, for the treatment of cancer has been an area of growing interest. Curcumin in its free form is poorly absorbed in the gastrointestinal tract and therefore may be limited in its clinical efficacy. Liposome encapsulation of this compound would allow systemic administration. The current study evaluated the preclinical antitumor activity of liposomal curcumin in colorectal cancer. We also compared the efficacy of liposomal curcumin with oxaliplatin, a standard chemotherapy for this malignancy. In vitro treatment with liposomal curcumin induced a dose-dependent growth inhibition [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt] and apoptosis [poly(ADP-ribose) polymerase] in the two human colorectal cancer cell lines tested (LoVo and Colo205 cells). There was also synergism between liposomal curcumin and oxaliplatin at a ratio of 4:1 in LoVo cells in vitro. In vivo, significant tumor growth inhibition was observed in Colo205 and LoVo xenografts, and the growth inhibition by liposomal curcumin was greater than that for oxaliplatin (P < 0.05) in Colo205 cells. Tumors from animals treated with liposomal curcumin showed an antiangiogenic effect, including attenuation of CD31 (an endothelial marker), vascular endothelial growth factor, and interleukin-8 expression by immunohistochemistry. This study establishes the comparable or greater growth-inhibitory and apoptotic effects of liposomal curcumin with oxaliplatin both in vitro and in vivo in colorectal cancer. We are currently developing liposomal curcumin for introduction into the clinical setting. [Mol Cancer Ther 2007;6(4):1276–82]
Luteolin sensitizes the anticancer effect of cisplatin via c-Jun NH2-terminal kinase–mediated p53 phosphorylation and stabilization
Authors: Shi Ranxin , Huang Qing , Zhu Xinqiang , Ong Yeong-Bing , Zhao Bin , Lu Jia , Ong Choon-Nam , Shen Han-Ming
Published: April 2007

Luteolin is an important flavonoid with a potential anticancer effect. In this study, we examined the molecular mechanisms involved in the sensitization effect of luteolin on cancer cell killing induced by cisplatin, an important cancer chemotherapeutic agent. First, we provided evidence that the sensitization effect of luteolin on cisplatin-induced apoptosis is p53 dependent, as such effect is only found in p53 wild-type cancer cells but not in p53 mutant cancer cells. Moreover, knockdown of p53 by small interfering RNA made p53 wild-type cancer cells resistant to luteolin and cisplatin. Second, we observed a significant increase of p53 protein level in luteolin-treated cancer cells without increase of p53 mRNA level, indicating the possible effect of luteolin on p53 posttranscriptional regulation. Third, we identified the critical role of c-Jun NH2-terminal kinase (JNK) in regulation of p53 protein stability: luteolin activates JNK, and JNK then stabilizes p53 via phosphorylation, leading to reduced ubiquitination and proteasomal degradation. Finally, by using an in vivo nude mice xenograft model, we confirmed that luteolin enhanced the cancer therapeutic activity of cisplatin via p53 stabilization and accumulation. In summary, data from this study reveal a novel molecular mechanism involved in the anticancer effect of luteolin and support its potential clinical application as a chemosensitizer in cancer therapy. [Mol Cancer Ther 2007;6(4):1338–47]
Uncommon tumors and exceptional therapies: paradox or paradigm?
Authors: Braiteh Fadi , Kurzrock Razelle
Published: April 2007

Why does it seem that, repeatedly, when a new treatment with a striking effect is discovered in the cancer field, it is effective for a very rare cancer type? For example, groundbreaking therapeutic discoveries have been made for extremely uncommon malignancies such as hairy cell leukemia, chronic myelogenous leukemia, seminoma, gastrointestinal stromal tumor, (del)5q myelodysplastic syndrome, and acute promyelocytic leukemia. In contrast, progress in the most common and most intensively studied tumors—lung, breast, prostate, and colon cancer—has been slow and incremental. We hypothesize that the reason for this phenomenon is that the pathophysiologic basis for a tumor being rare is one and the same as the reason that it may ultimately be so treatable. That is, if a cancer can be derived only via a single aberrant molecular genetic aberration, then it should be both rare and easily targeted by a molecular cancer therapeutic approach. If, on the other hand, many distinct pathways can lead to the development of a specific tumor type, it should occur much more commonly and be significantly more difficult to treat. The corollary to our hypothesis is the prediction that new therapies will continue to show their most salutary effects in rare cancers. Furthermore, only by stratifying the common tumors, especially when using targeted agents, into the molecular subsets of diseases that compose them are we likely to achieve a substantial effect in these disorders. [Mol Cancer Ther 2007;6(4):1175–9]
Loss of ataxia telangiectasia mutated– and Rad3-related function potentiates the effects of chemotherapeutic drugs on cancer cell survival
Authors: Wilsker Deborah , Bunz Fred
Published: April 2007

The diverse responses of human cells to various forms of DNA damage are controlled by a complex network of signaling proteins. There has been considerable interest in the components of this signaling apparatus as potential targets for new forms of anticancer therapy. In this report, we examine the contributions of an upstream signaling molecule, the ataxia telangiectasia mutated– and Rad3-related (ATR) protein kinase, to the resistance of cancer cells to DNA-damaging agents that are commonly used as anticancer therapeutics. Loss of ATR function in knock-in cancer cells strikingly enhanced the effects of several of the most commonly used therapeutic compounds, impeding the progression of the cell cycle and reducing long-term cancer cell survival. Loss of ATR function potentiated the toxicity of alkylating agents most strikingly, antimetabolites moderately, and double-strand break–inducing agents to a lesser extent. These results suggest that specific inhibition of ATR activity will be a valid strategy to increase the effectiveness of currently used modes of therapy. [Mol Cancer Ther 2007;6(4):1406–13]
Loss of mitochondrial membrane potential is inhibited by bombesin in etoposide-induced apoptosis in PC-3 prostate carcinoma cells
Authors: Salido Mercedes , Gonzalez Juan L. , Vilches Jose
Published: April 2007

Neuroendocrine secretory products and their interactions with epithelial prostate cells are currently under investigation in order to understand their significance in the pathogenesis, prognosis, and therapy of prostate carcinoma. These neuropeptides have the potential to disrupt the balance between cell death and cell growth in the tumor. Our research was based on the role of bombesin in modulating the mitochondrial membrane potential (Δψm) in cell death induced by etoposide on PC-3 cells. Cells were cultured and stained with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). At low membrane potentials, JC-1 produces a green fluorescence, and at high membrane potentials, it forms “J aggregates” with red fluorescence. Cells were examined in a confocal microscope. For quantitative analyses, regions of interest were selected. The size, number of pixels, and ratios between fluorescence intensity in the red and green channels in each region of interest were calculated. The loss of Δψm in etoposide-treated PC-3 cells was prevented by bombesin. The quantitative analysis of JC-1–stained cells revealed a significant decrease in the red (high Δψm) to green (low Δψm) ratio in etoposide-treated cells when compared with control cells, which was restored in the presence of bombesin (P < 0.00001). The interaction between treatments and area (P = 0.0002) was highly significant, and confirms that PC-3 cells keep their apoptosis machinery, showing an apoptotic volume decrease in response to etoposide. The protection by bombesin occurs by inhibition of apoptosis and maintenance of mitochondrial integrity. New therapeutic protocols and trials need to be developed to test drugs acting through the neutralization of antiapoptotic intracellular pathways mediated by neuroendocrine hormones. [Mol Cancer Ther 2007;6(4):1292–9]
Clofibric acid, a peroxisome proliferator–activated receptor α ligand, inhibits growth of human ovarian cancer
Authors: Yokoyama Yoshihito , Xin Bing , Shigeto Tatsuhiko , Umemoto Mika , Kasai-Sakamoto Akiko , Futagami Masayuki , Tsuchida Shigeki , Al-Mulla Fahd , Mizunuma Hideki
Published: April 2007

Recent reports have shown that peroxisome proliferator–activated receptor (PPAR)α ligands reduce growth of some types of malignant tumors and prevent carcinogenesis. In this study, we investigated the inhibitory effect of clofibric acid (CA), a ligand for PPARα on growth of ovarian malignancy, in in vivo and in vitro experiments using OVCAR-3 and DISS cells derived from human ovarian cancer and aimed to elucidate the molecular mechanism of its antitumor effect. CA treatment significantly suppressed the growth of OVCAR-3 tumors xenotransplanted s.c. and significantly prolonged the survival of mice with malignant ascites derived from DISS cells as compared with control. CA also dose-dependently inhibited cell proliferation of cultured cell lines. CA treatment increased the expression of carbonyl reductase (CR), which promotes the conversion of prostaglandin E2 (PGE2) to PGF2α, in implanted OVCAR-3 tumors as well as cultured cells. CA treatment decreased PGE2 level as well as vascular endothelial growth factor (VEGF) amount in both of OVCAR-3–tumor and DISS-derived ascites. Reduced microvessel density and induced apoptosis were found in solid OVCAR-3 tumors treated by CA. Transfection of CR expression vector into mouse ovarian cancer cells showed significant reduction of PGE2 level as well as VEGF expression. These results indicate that CA produces potent antitumor effects against ovarian cancer in conjunction with a reduction of angiogenesis and induction of apoptosis. We conclude that CA could be an effective agent in ovarian cancer and should be tested alone and in combination with other anticancer drugs. [Mol Cancer Ther 2007;6(4):1379–86]