Effects of repetitive transcranial magnetic stimulation on cerebral glucose metabolism.

OBJECTIVE: To investigate the mechanisms underlying the effect of repetitive transcranial magnetic stimulation (rTMS) on post-stroke hemiplegia, we assessed alterations in cerebral glucose metabolism. METHODS: Five post-stroke hemiplegic patients (three targeted for upper limb impairment and two targeted for lower limb impairment) aged 62.6 ± 6.1 years (mean ± standard deviation) with a duration since stroke onset of 3.5 ± 3.8 years participated in this preliminary study. Cerebral glucose metabolism was measured twice-before and after rTMS with intensive rehabilitation-using positron emission tomography with [18F]fluorodeoxyglucose. The Asymmetry Index (AI) was calculated to assess laterality of metabolism between the lesional and contralesional motor areas. The alteration rates of AI (%ΔAI) were compared between participants in whom rTMS was effective and ineffective. RESULTS: Two of the three upper-limb-targeted patients and one of the two lower-limb-targeted patients showed motor function improvements following rTMS treatment. All three patients who responded to rTMS had improved laterality of cerebral glucose metabolism in motor areas, commonly in the precentral gyrus, with an %ΔAI of approximately 10%. In contrast, the two patients who did not respond to rTMS had no improvements in laterality. CONCLUSIONS: These results suggest for the first time that improved glucose metabolism is associated with improved motor function after a combination of rTMS and intensive rehabilitation.

[End-of-Life Care at Nursing Facilities].

In Japan, it has been more common for elderly people to die in a hospital than at home. However, recently, increasingly more elderly people want to die where they have been living for a long time, such as their homes or nursing care facilities. We have been visiting patients at various types of nursing care facilities for ten years, including terminal care services followed by confirming death for 152 patients. Our study showed that 87 patients died of old age, which was the top cause of death. We also found that nursing care facilities, where nurses work full-time, provided terminal care for the greatest number of residents, which was 124 patients. The average period between admission to a facility and death was 3 years 3 months. The number of patients who receive terminal nursing care at a facility is increasing annually. The number of elderly people who die at home or a nursing care facility, rather than a hospital, will keep increasing in the near future.

[Association of the Types of Dementia and the Incidence of Hypertension, Diabetes Mellitus, or Bone Fracture].

Patients with dementia tend to have other chronic diseases, such as hypertension or diabetes mellitus. As dementia progresses, patients tend to decline to a frail state, resulting in bone fracture. In this study, we examined the relationship of the types of dementia and the incidence of other diseases and bone fracture.

[Revision of Medical Fee System for Treatment at Nursing Homes - The Influential Consideration of the Introduction of One-Patient-Per-Visit Method on the Welfare of Elderly Patients at Nursing Homes].

The 2014 revision of the medical fee system includes the introduction of a one-patient-per-visit method at nursing homes, which should be followed to avoid a drastic reduction in medical fees. We followed the new method, resulting in much more frequent visits to nursing homes(For example, we visit a facilitythree times per week instead of the previous two times per month). Frequent visits to multiple facilities are time- and effort-consuming on our side as a clinic, but, on the other hand, patients have more opportunities to see a doctor when theyare sick even if theyare not scheduled to do so. In this study, we examined how the new method affects the welfare of elderlypatients at nursing homes.

The role of autophagy in cancer development and response to therapy.

Autophagy is a process in which subcellular membranes undergo dynamic morphological changes that lead to the degradation of cellular proteins and cytoplasmic organelles. This process is an important cellular response to stress or starvation. Many studies have shed light on the importance of autophagy in cancer, but it is still unclear whether autophagy suppresses tumorigenesis or provides cancer cells with a rescue mechanism under unfavourable conditions. What is the present state of our knowledge about the role of autophagy in cancer development, and in response to therapy? And how can the autophagic process be manipulated to improve anticancer therapeutics?

[Assessment of the association between nursing care services, hypertension, and Alzheimer's disease in elderly patients with late-stage diabetes].

In an aging society with fewer children, diabetes self-control is difficult for elderly patients. Under these circumstances, it is expected that living in care homes for the elderly and institutions where nursing care services could be provided will help improve the prognosis of diabetic patients. Therefore, we assessed whether HbA(1c). levels (National Glycohemoglobin Standardization Program : NGSP) in 121 elderly patients with late-stage diabetes receiving home medical care in our clinic from March 2008 to March 2013 improved with nursing care services.

[Association of anemia with nutrition in elderly patients].

We analyzed the data for 59 cases in which elderly patients receiving home medical care presented with anemia. Our results demonstrated that the cause of anemia was iron deficiency, chronic inflammation, or chronic kidney disease in 75% of the patients, and that anemia was improved in more than 80% of the patients after appropriate therapeutic treatment. We also found so-called senile anemia with no particular causes in 20% of the patients, and in some of them, both nutrition and anemia have improved. We concluded that not only the treatment of anemia, but also the improvement of nutrition, is important in elderly patients with anemia.

[Assessment of the relationships between insomnia and hypnotic use with the risk of falls and bone fractures in late-stage elderly patients].

To determine whether insomnia and hypnotic use are predictors of falls and bone fractures in older people, we assessed their relationship using clinical data for 599 late-stage elderly patients receiving home medical care in our clinic from May 2008 to December 2011. Our analysis demonstrated that hypnotic use is not associated with a greater risk of subsequent falls and bone fractures.

The tumor suppressor gene ARHI regulates autophagy and tumor dormancy in human ovarian cancer cells.

The role of autophagy in oncogenesis remains ambiguous, and mechanisms that induce autophagy and regulate its outcome in human cancers are poorly understood. The maternally imprinted Ras-related tumor suppressor gene aplasia Ras homolog member I (ARHI; also known as DIRAS3) is downregulated in more than 60% of ovarian cancers, and here we show that re-expression of ARHI in multiple human ovarian cancer cell lines induces autophagy by blocking PI3K signaling and inhibiting mammalian target of rapamycin (mTOR), upregulating ATG4, and colocalizing with cleaved microtubule-associated protein light chain 3 (LC3) in autophagosomes. Furthermore, ARHI is required for spontaneous and rapamycin-induced autophagy in normal and malignant cells. Although ARHI re-expression led to autophagic cell death when SKOv3 ovarian cancer cells were grown in culture, it enabled the cells to remain dormant when they were grown in mice as xenografts. When ARHI levels were reduced in dormant cells, xenografts grew rapidly. However, inhibition of ARHI-induced autophagy with chloroquine dramatically reduced regrowth of xenografted tumors upon reduction of ARHI levels, suggesting that autophagy contributed to the survival of dormant cells. Further analysis revealed that autophagic cell death was reduced when cultured human ovarian cancer cells in which ARHI had been re-expressed were treated with growth factors (IGF-1, M-CSF), angiogenic factors (VEGF, IL-8), and matrix proteins found in xenografts. Thus, ARHI can induce autophagic cell death, but can also promote tumor dormancy in the presence of factors that promote survival in the cancer microenvironment.

[Management of elderly patients with high fever].

We analyzed 102 cases of data in which elderly patients with high fever received a home medical care. Our results demonstrated that 68 cases recovered within several days after taking symptom-relieving drugs and antibiotics. Thirty four cases were referred to a hospital for examinations; 25 cases resulted in admission to a hospital for further treatments. When we take care of the elderly patients with high fever, it is imperative to carefully observe the conditions of the patients and refer to a hospital in a timely and adequate manner.

[Usefulness of NT-proBNP as a heart disease marker for late-stage elderly patients receiving home medical care].

The level of N-terminal pro-brain natriuretic peptide(NT-proBNP)is a strong predictor of mortality among patients with coronary heart disease, and may be a strong prognostic marker for late-stage elderly patients with heart disease receiving home medical care. We assessed the relationshipbetween NT-proBNP levels and clinical data of 240 late-stage elderly patients receiving home medical care in our clinic from May 2008 to February 2011.

Tissue transglutaminase inhibits autophagy in pancreatic cancer cells.

Elevated expression of tissue transglutaminase (TG2) in cancer cells has been implicated in the development of drug resistance and metastatic phenotypes. However, the role and the mechanisms that regulate TG2 expression remain elusive. Here, we provide evidence that protein kinase Cdelta (PKCdelta) regulates TG2 expression, which in turn inhibits autophagy, a type II programmed cell death, in pancreatic cancer cells that are frequently insensitive to standard chemotherapeutic agents. Rottlerin, a PKCdelta-specific inhibitor, and PKCdelta small interfering RNA (siRNA) down-regulated the expression of TG2 mRNA and protein and induced growth inhibition without inducing apoptosis in pancreatic cancer cells. Inhibition of PKCdelta by rottlerin or knockdown of TG2 protein by a TG2-specific siRNA resulted in a marked increase in autophagy shown by presence of autophagic vacuoles in the cytoplasm, formation of the acidic vesicular organelles, membrane association of microtubule-associated protein 1 light chain 3 (LC3) with autophagosomes, and a marked induction of LC3-II protein, important hallmarks of autophagy, and by electron microscopy. Furthermore, inhibition of TG2 by rottlerin or by the siRNA led to accumulation of green fluorescent protein (GFP)-LC3-II in autophagosomes in pancreatic cancer cells transfected with GFP-LC3 (GFP-ATG8) expression vector. Knockdown of Beclin-1, a specific autophagy-promoting protein and the product of Becn1 (ATG6), inhibited rottlerin-induced and TG2 siRNA-induced autophagy, indicating that Beclin-1 is required for this process. These results revealed that PKCdelta plays a critical role in the expression of TG2, which in turn regulates autophagy. In conclusion, these results suggest a novel mechanism of regulation of TG2 and TG2-mediated autophagy in pancreatic cancer cells.

Telomere 3' overhang-specific DNA oligonucleotides induce autophagy in malignant glioma cells.

Telomere 3' overhang-specific DNA oligonucleotides (T-oligos) induce cell death in cancer cells, presumably by mimicking telomere loop disruption. Therefore, T-oligos are considered an exciting new therapeutic strategy. The purpose of this study was to elucidate how T-oligos exert antitumor effects on human malignant glioma cells in vitro and in vivo. We demonstrated that T-oligos inhibited the proliferation of malignant glioma cells through induction of nonapoptotic cell death and mitochondria hyperpolarization, whereas normal astrocytes were resistant to T-oligos. Tumor cells treated with T-oligos developed features compatible with autophagy, with development of autophagic vacuoles and conversion of an autophagy-related protein, microtubule-associated protein 1 light chain 3 from type I (cytoplasmic form) to type II (membrane form of autophagic vacuoles). A reverse-phase protein microarray analysis and Western blotting revealed that treatment with T-oligos inhibited the mammalian target of the rapamycin (mTOR) and the signal transducer and activator of transcription 3 (STAT3). Moreover, pretreatment with T-oligos significantly prolonged the survival time of mice inoculated intracranially with malignant glioma cells compared with that of untreated mice and those treated with control oligonucleotides (P=0.0065 and P=0.043, respectively). These results indicate that T-oligos stimulate the induction of nonapoptotic autophagic also known as type II programmed cell death and are thus promising in the treatment of malignant glioma.

Phosphorylated Pak1 level in the cytoplasm correlates with shorter survival time in patients with glioblastoma.

PURPOSE: Glioblastoma is the most common primary malignant tumor in the brain. It aggressively invades the surrounding parenchyma, often allowing the tumor to progress after surgery. Accumulating evidence has shown that phosphorylated p21-activated kinase 1 (Pak1), a mediator of small guanosine triphosphatases, plays a role in the proliferation, survival, and invasiveness of cancer cells. Thus, we examined patterns of Pak1 expression in glioblastoma and sought to determine whether the level of phosphorylated Pak1 in glioblastoma cells is associated with patient survival time. EXPERIMENTAL DESIGN: We carried out immunohistochemical staining for phosphorylated Pak1 in tumor specimens from 136 patients with glioblastoma; the tumors were classified according to Pak1 protein levels in the cytoplasm and nucleus. We compared the patients' overall survival times using Kaplan-Meier analysis and estimated the effects of levels of cytoplasmic or nuclear phosphorylated Pak1. We then down-regulated Pak1 by using small interfering RNA to knock down Pak1 in two glioblastoma cell lines to determine whether Pak1 contributed to cell viability and invasion. RESULTS: Median overall survival was significantly shorter in patients with tumors showing a moderate or high level of cytoplasmic phosphorylated Pak1 than in patients with tumors showing no cytoplasmic phosphorylated Pak1. The level of nuclear phosphorylated Pak1 was not related to survival time. Knockdown of Pak1 suppressed the invasion, but not the viability, of U87-MG and U373-MG cells. CONCLUSIONS: The presence of phosphorylated Pak1 in the cytoplasm of glioblastoma cells is associated with shorter survival, and Pak1 plays a role in the invasiveness of glioblastoma. These data suggest that Pak1 might be a potential target for the management of glioblastoma.

Eupalmerin acetate, a novel anticancer agent from Caribbean gorgonian octocorals, induces apoptosis in malignant glioma cells via the c-Jun NH2-terminal kinase pathway.

The marine ecosystem is a vast but largely untapped resource for potential naturally based medicines. We tested 15 compounds derived from organisms found in the Caribbean Sea (14 gorgonian octocoral-derived compounds and one sponge-derived compound) for their anticancer effects on human malignant glioma U87-MG and U373-MG cells. Eupalmerin acetate (EPA) was chosen as the lead compound based on its longer-term stability and greater cytotoxicity than those of the other compounds we tested in these cell types. EPA induced G(2)-M cell cycle arrest and apoptosis via the mitochondrial pathway; it translocated Bax from the cytoplasm to the mitochondria and dissipated the mitochondrial transmembrane potential in both cell types. EPA was found to increase phosphorylated c-Jun NH(2)-terminal kinase (JNK) by >50% in both U87-MG and U373-MG cells. A specific JNK inhibitor, SP600125, inhibited EPA-induced apoptosis, confirming the involvement of the JNK pathway in EPA-induced apoptotic cell death. Furthermore, 7 days of daily intratumoral injections of EPA significantly suppressed the growth of s.c. malignant glioma xenografts (P < 0.01, on day 19). These results indicate that EPA is therapeutically effective against malignant glioma cells in vitro and in vivo and that it, or a similar marine-based compound, may hold promise as a clinical anticancer agent.

Combined effect of 2-5A-linked antisense against telomerase RNA and conventional therapies on human malignant glioma cells in vitro and in vivo.

We recently showed that therapy with 2'-5'-oligoadenylate (2-5A)-linked antisense against human telomerase RNA component (2-5A-anti-hTR) is a novel telomerase-targeting strategy against malignant gliomas. In this study, we investigated conventional chemotherapeutic agents and gamma-irradiation (IR) to determine whether they could augment the efficacy of 2-5A-anti-hTR against these tumors in vitro and in vivo. Treatment with 2-5A-anti-hTR inhibited the viability of U373-MG and U87-MG malignant glioma cells in a dose-dependent manner; the antitumor effect resulted from induction of apoptosis. Also, telomerase-positive astrocytes with oncogenic Ras were more sensitive to 2-5A-anti-hTR than were those without oncogenic Ras. In addition, we sought to determine the combined effect of 2-5A-anti-hTR with N, N'-bis (2-chloroethyl)-N-nitrosourea (BCNU), cisplatin (CDDP), paclitaxel (PTX), temozolomide (TMZ), or IR. When we administered the combination treatments on the same day, PTX and IR showed a greater combined effect with 2-5A-anti-hTR on both tumor cell lines than did BCNU, CDDP and TMZ. However, all of the combination regimens were synergistic when we first treated tumor cells with 2-5A-anti-hTR for 24 h and then exposed them to the conventional treatments. Apoptosis-inducing agents (CDDP and PTX) but not autophagy-inducing therapies (TMZ and IR) enhanced the incidence of apoptosis caused by 2-5A-anti-hTR. Lastly, we observed a combinatorial effect of 2-5A-anti-hTR and TMZ in vivo in subcutaneous U87-MG tumors in nude mice. Interestingly, treatment with TMZ increased the incidence of apoptosis in subcutaneous tumor cells treated with 2-5A-anti-hTR. These results suggest that 2-5A-anti-hTR is preferable in combination with established cancer therapies.

Akt inhibitor shows anticancer and radiosensitizing effects in malignant glioma cells by inducing autophagy.

Autophagy, or programmed cell death type II, is one of the responses of cancer cells to various therapies, including ionizing radiation. Recently, we have shown that radiation induces autophagy, but not apoptosis, in various malignant glioma cell lines. Autophagy is mainly regulated by the mammalian target of rapamycin (mTOR) pathway. The Akt/mTOR pathway also mediates oncogenesis and radioresistance. Thus, we hypothesized that inhibiting this pathway has both an anticancer and radiosensitizing effect by activating autophagy. The purpose of our study was therefore to determine whether and by which mechanisms an Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate, had anticancer and radiosensitizing effects on malignant glioma U87-MG and radioresistant U87-MG cells with a consistitutively active form of epidermal growth factor receptor (U87-MGDeltaEGFR). Treatment with the Akt inhibitor successfully inhibited Akt activity and reduced cell viability in both cell lines. In terms of the mechanism, the Akt inhibitor decreased phosphorylated p70S6 kinase, a downstream target of Akt, and induced autophagy, but not apoptosis. Furthermore, the Akt inhibitor radiosensitized both U87-MG and U87-MGDeltaEGFR cells by enhancing autophagy. Specific inhibition of Akt using the dominant-negative Akt plasmid also resulted in enhanced radiation-induced autophagy. In conclusion, an Akt inhibitor showed anticancer and radiosensitizing effect on U87-MG and U87-MGDeltaEGFR cells by inducing autophagy. Thus, Akt inhibitors may represent a promising new therapy as a single treatment or used in combination with radiation for malignant gliomas, including radioresistant ones that express DeltaEGFR.

Telomerase RNA inhibition using antisense oligonucleotide against human telomerase RNA linked to a 2',5'-oligoadenylate.

Telomerase, a ribonucleoprotein enzyme, is detected in the vast majority of cancers, including malignant gliomas, but not in most normal somatic cells. To inhibit telomerase function effectively, we have adopted the 2',5'-oligoadenylate (2-5A) antisense system. 2-5A is a mediator of one pathway of interferon actions by activating RNase L, resulting in single-stranded RNA cleavage. By linking 2-5A to an antisense oligonucleotide, RNase L degrades the targeted RNA specifically and effectively. Therefore, we have synthesized the antisense oligonucleotide against human telomerase RNA component (hTR) linked to 2-5A (2-5A-anti-hTR) and have demonstrated its antitumor effect on telomerase-positive cancer cells in vitro and in vivo.

Autophagic cell death of human pancreatic tumor cells mediated by oleandrin, a lipid-soluble cardiac glycoside.

Lipid-soluble cardiac glycosides such as bufalin, oleandrin, and digitoxin have been suggested as potent agents that might be useful as anticancer agents. Past research with oleandrin, a principle cardiac glycoside in Nerium oleander L. (Apocynaceae), has been shown to induce cell death through induction of apoptosis. In PANC-1 cells, a human pancreatic cancer cell line, cell death occurs not through apoptosis but rather through autophagy. Oleandrin at low nanomolar concentrations potently inhibited cell proliferation associated with induction of a profound G(2)/M cell cycle arrest. Inhibition of cell cycle was not accompanied by any significant sub G1 accumulation of cells, suggesting a nonapoptotic mechanism. Oleandrin-treated cells exhibited time- and concentration-dependent staining with acridine orange, a lysosomal stain. Subcellular changes within PANC-1 cells included mitochondrial condensation and translocation to a perinuclear position accompanied by vacuoles. Use of a fluorescent oleandrin analog (BODIPY-oleandrin) revealed co-localization of the drug within cell mitochondria. Damaged mitochondria were found within autophagosome structures. Formation of autophagosomes was confirmed through electron microscopy and detection of green fluorescent protein-labeled light chain 3 association with autophagosome membranes. Also observed was a drug-mediated inhibition of pAkt formation and up-regulation of pERK. Transfection of Akt into PANC-1 cells or inhibition of pERK activation by MAPK inhibitor abrogated oleandrin-mediated inhibition of cell growth, suggesting that the reduction of pAkt and increased pERK are important to oleandrin's ability to inhibit tumor cell proliferation. The data provide insight into the mechanisms and role of a potent, lipid-soluble cardiac glycoside (oleandrin) in control of human pancreatic cancer proliferation.

Inhibition of the DNA-dependent protein kinase catalytic subunit radiosensitizes malignant glioma cells by inducing autophagy.

DNA-dependent protein kinase (DNA-PK) plays a major role in the repair of DNA double-strand breaks induced by ionizing radiation (IR). Lack of DNA-PK causes defective DNA double-strand break repair and radiosensitization. In general, the cell death induced by IR is considered to be apoptotic. On the other hand, nonapoptotic cell death, autophagy, has recently attracted attention as a novel response of cancer cells to chemotherapy and IR. Autophagy is a protein degradation system characterized by a prominent formation of double-membrane vesicles in the cytoplasm. Little is known, however, regarding the relationship between DNA-PK and IR-induced autophagy. In the present study, we used human malignant glioma M059J and M059K cells to investigate the role of DNA-PK in IR-induced apoptotic and autophagic cell death. Low-dose IR induced massive autophagic cell death in M059J cells that lack the catalytic subunit of DNA-PK (DNA-PKcs). Most M059K cells, the counterpart of M059J cells in which DNA-PKcs are expressed at normal levels, survived, and proliferated although a small portion of the cells underwent apoptosis. Low-dose IR inhibited the phosphorylation of p70(S6K), a molecule downstream of the mammalian target of rapamycin associated with autophagy in M059J cells but not in M059K cells. The treatment of M059K cells with antisense oligonucleotides against DNA-PKcs caused radiation-induced autophagy and radiosensitized the cells. Furthermore, antisense oligonucleotides against DNA-PKcs radiosensitized other malignant glioma cell lines with DNA-PK activity, U373-MG and T98G, by inducing autophagy. The specific inhibition of DNA-PKcs may be promising as a new therapy to radiosensitize malignant glioma cells by inducing autophagy.