A novel inhibitor of the STAT3 pathway induces apoptosis in malignant glioma cells both in vitro and in vivo.

Signal transducer and activator of transcription-3 (STAT3) is constitutively activated in a variety of cancer types, including malignant gliomas. STAT3 is activated by phosphorylation of a tyrosine residue, after which it dimerizes and translocates into the nucleus. There it regulates the expression of several genes responsible for proliferation and survival at the transcriptional level. A selective inhibitor of STAT3 phosphorylation, AG490, has been shown to inhibit growth and induce apoptosis in some cancer cell types. However, although AG490 routinely shows in vitro anticancer activity, it has not consistently demonstrated an in vivo anticancer effect in animal models. Here, we have tested WP1066, a novel inhibitor structurally related to AG490 but significantly more potent and active, against human malignant glioma U87-MG and U373-MG cells in vitro and in vivo. IC(50) values for WP1066 were 5.6 muM in U87-MG cells and 3.7 muM in U373-MG cells, which represents 18-fold and eightfold increases in potency, respectively, over that of AG490. WP1066 activated Bax, suppressed the expression of c-myc, Bcl-X(L) and Mcl-1, and induced apoptosis. Systemic intraperitoneal administration of WP1066 in mice significantly (P<0.001) inhibited the growth of subcutaneous malignant glioma xenografts during the 30-day follow-up period. Immunohistochemical analysis of the excised tumors revealed that phosphorylated STAT3 levels in the WP1066 treatment group remained inhibited at 3 weeks after the final WP1066 injection, whereas tumors from the control group expressed high levels of phosphorylated STAT3. We conclude that WP1066 holds promise as a therapeutic agent against malignant gliomas.

Silencing mammalian target of rapamycin signaling by small interfering RNA enhances rapamycin-induced autophagy in malignant glioma cells.

The mammalian target of rapamycin (mTOR) plays a central role in regulating the proliferation of malignant glioma cells, and mTOR-specific inhibitors such as rapamycin analogs are considered as promising therapy for malignant gliomas. However, the efficacy of mTOR inhibitors alone in the treatment of patients with malignant gliomas is only modest, potentially because these agents rather than acting as mTOR kinase inhibitors instead interfere with the function of only mTOR/raptor (regulatory-associated protein of mTOR) complex and thus do not perturb all mTOR functions. The purpose of this study was to determine whether global inhibition of the mTOR molecule enhances the antitumor effect of rapamycin on malignant glioma cells. We showed that rapamycin induced autophagy and that inhibition of autophagy by small interfering RNA (siRNA) directed against autophagy-related gene Beclin 1 attenuated the cytotoxicity of rapamycin in rapamycin-sensitive tumor cells, indicating that the autophagy was a primary mediator of rapamycin's antitumor effect rather than a protective response. Exogenous expression of an mTOR mutant interfering with its kinase activity markedly enhanced the incidence of rapamycin-induced autophagy. Moreover, silencing of mTOR with siRNA augmented the inhibitory effect of rapamycin on tumor cell viability by stimulating autophagy. Importantly, not only rapamycin-sensitive malignant glioma cells with PTEN mutations but also rapamycin-resistant malignant glioma cells with wild-type PTEN were sensitized to rapamycin by mTOR siRNA. These results indicate that rapamycin-induced autophagy is one of the agent's antitumor effects and that silencing or inhibiting mTOR kinase activity could enhance the effectiveness of rapamycin.

Ionizing radiation induces apoptosis and inhibits neuronal differentiation in rat neural stem cells via the c-Jun NH2-terminal kinase (JNK) pathway.

A substantial number of neural stem cells (NSCs) continue to proliferate and generate neurons in the central nervous system throughout life. Ionizing radiation, an important adjuvant therapy for glioma patients, may damage NSCs and cause neuronal deficits, such as cognitive dysfunction and memory impairment. However, the precise mechanism of radiation effects on death and differentiation of NSCs remains largely unknown. Here, we found that radiation induced apoptosis in NSCs via the mitochondrial pathway, upregulating the ratio of Bax to Bcl-2 and releasing cytochrome c into the cytoplasm. Radiation also inhibited neuronal differentiation of NSCs by 50%. Of the three stress-associated mitogen-activated protein kinases (MAPKs), only c-Jun NH(2)-terminal kinase (JNK) was activated in NSCs after radiation. Interestingly, JNK inhibition by the specific inhibitor SP600125 rescued NSCs from apoptosis and improved neuronal differentiation. Furthermore, we examined whether radiation directly inhibits neuronal differentiation or not. Radiation did not affect the promoter activity of NeuroD, a basic helix-loop-helix transcription factor that regulates the expression of neuronal differentiation markers. Radiation induced more apoptosis in NeuroD-positive cells than NeuroD-negative cells. We concluded that radiation activates JNK and induces apoptosis, especially in neural progenitor cells, resulting in the inhibition of neurogenesis. Our findings raise the possibility that JNK inhibition has therapeutic potential in protecting NSCs from the adverse effects of radiation.

Measurement of tumor blood flow using colored dye extraction microspheres in two rat tumor models.

A technique that can measure tumor blood flow easily, accurately and economically is required to study tumor angiogenesis and angiogenesis inhibition. Using dye extraction colored microspheres, we measured tumor blood flow in Sato lung carcinoma (SLC) and ascites hepatoma LY80 in rats. Colored microspheres were infused into tumor-bearing rats via a catheter in the left ventricle. After removal of the tumor and the liver, the tissue samples were dissolved, and the microspheres were isolated. Dye was extracted, and the dye concentration was quantified by spectrophotometry. The dye concentration per gram of tumor was compared with that per gram of liver as follows (AU = absorbency units): [AU per gram of tumor] / [AU per gram of liver] X 100 = (%). Tumor blood flow corrected for wet weight was calculated as follows: [blood flow to tumor] = [AU per gram of tumor] X [reference withdrawal rate] / [AU per gram of reference blood]. Tumor blood flow rate was divided by tumor weight to yield ml. min-1g-1. The tumors were also examined histologically, and casts of the tumor vasculature were prepared with silicone rubber. Blood flow 2 weeks after transplantation was equivalent to 1/10 and 1/2 at 1 week in SLC and LY80 tumors, respectively (SLC, P=0.009, n=10; LY80, P=0.05, n=10). These decreases in tumor blood flow were associated with underlying pathological and vascular change. Blood flow in LY80 tumors negatively correlated with tumor volume (P=0.009, n=10). We concluded that the colored microsphere method, initially developed to measure organ blood flow, is also useful for estimating tumor blood flow in rats.

[A role of visiting nurses in providing the terminal patients with their desirable life-discussion regarding the place of death of the patients who were registered for visiting nurse system].

AIM: To evaluate the prognosis of the patients who had visiting nurse service and discuss the place of death (life at the terminal stage). To determine the roles of visiting nurses in providing the patients at a terminal stage with their desirable life till death. METHODS: A total of 180 patients, who were registered for their home healthcare service in our Shonan Kamakura General Hospital and died between January 2000 and February 2001, were subjected to the study. All the subjects were classified into 3 groups according to the places of their death, 1) death at home, 2) death in the hospital and 3) death upon arrival after the admission to the hospital. Moreover, the following items were also surveyed and analyzed: 1) diagnosis (name of diseases), 2) cause of death, 3) age, 4) family structure, 5) whether their primary care physicians explained the prognosis and possible expected conditions to the patients and their family before hand, and 6) how the visiting nurses interact with the patients and their family members. RESULTS: Sixty-six patients died at home, 105 in the hospital and 9 upon arrival at the hospital. During this survey period, there were a total of 5,274 and 5,574 visits by primary care physicians and visiting nurses, respectively. The patients who died at home were more often observed in the patients whose primary care physicians explained their conditions to them and whose visiting nurses closely related to them. Moreover, the patients with malignant tumor also more often died at home. On the contrary, there were very few patients with chronic diseases, with whom death at home was accepted and agreed before hand, and there were some cases with chronic diseases who died inside of the ambulance transported on the way to the hospital after a sudden change in their conditions. DISCUSSION AND CONCLUSION: In order to have the patients live their desirable life till their death, it is required for the caretakers to prepare their mind for the day of the patient's death in addition to the patient's own wishes. For the patients with malignant tumor, it is easy to predict their prognosis, thus the caretakers can get prepared for the day of the patient's death. On the contrary, in case of the patients with chronic diseases, it is more difficult for the caretakers to experience an indefinite time with the patients since their prognosis is generally longer but the sudden change in their conditions may give the caretakers a high anxiety. Thus, it is essential for the visiting nurses to play a role as a mediator to interact between the patients and their family members, and their primary care physicians, and to establish a trustful relationship with the patients while their conditions are still stable. Moreover, similar to the malignant patients, the visiting nurses should explain the situations to the patients with chronic diseases, that they can choose the place of their own death and specific medical treatment at emergency and can decide the detail for their terminal stage with their family members. Thus, it was considered to be very important that the visiting nurses should frequently confirm these issues with the patients and their family according to their conditions.

[Clinical analysis in patients undergoing extended resection of pT3 non-small cell lung cancer].

Among pT3 cases there contain various subgroups in terms of the organ which is involved in. We analyzed medical records of 85 consecutive patients who underwent extended surgery with diagnosis of pT3 excluding interlober invasion. As regards to the site of invasion, there are not significant differences in survival between pleural invasion, chest wall involvement, pericardial invasion, and diaphragmatic invasion. However, survival of patients who showed involvement of main bronchus seemed better than other groups. Survival of pT3 cases are in part determined by lymph node involvement, N0 group showed 36.0% 5 year survival rate whereas N1 group 20.0%, and there are no patient with N2 disease who survived 5 years. Among pleural and chest wall involvement group, N0 group showed 34.2% 5 year survival and there are no survival in N1 and N2 group. As regards to histologic subgroups, there are not significant differences between each group. Thus we conclude that in pT3 cases, N0 cases are the best candidate for surgical resection, and that adjuvant therapy is necessary for those with N1 or N2 involvement. Cases with bronchial extension should not be argued in the same field of locally invasive lung cancer because of better survival.