Prolonged control of insulin-dependent diabetes via intramuscular expression of plasmid-encoded single-strand insulin analogue.

Daily insulin injection is necessary for the treatment of the insulin-dependent diabetes. However, the process is painful and inconvenient. Accordingly, we have made exploratory efforts to establish an alternative method for continuous insulin supply via intramuscular injection of a designed plasmid encoding the single-strand insulin analogue (SIA), which provides safe, effective and prolonged control of insulin-dependent diabetes. To generate a SIA, a short flexible peptide was alternatively introduced into the natural proinsulin to replace its original long and rigid C-peptide. Then, the synthetic promoter SP301 was used to drive potent and specific expression of SIA in skeletal muscle cells. By combining the Pluronic L64 and low-voltage electropulse (L/E), the specialized gene delivery technique was applied to efficiently transfer the constructed plasmid into skeletal muscle cells via intramuscular injection. Through these efforts, a plasmid-based intramuscular gene expression system was established and improved, making it applicable for gene therapy. The plasmid-expressed SIA showed biological functions that were similar to that of natural insulin. A single L/E-pSP301-SIA administration provided sustained SIA expression in vivo for about 1.5 months. In addition, the diabetic mice treated with L/E-pSP301-SIA were much healthier than those with other treatments. This plasmid-based system was safe for the treatment of diabetes and did not cause immune responses or pathological damage. The results confirmed that, in a mouse model, long-term positive effects were achieved by a single intramuscular L/E-pSP301-SIA injection, which consequently provided reliable experimental basis for its clinical application for the treatment of diabetes mellitus with promising prospects.

Glucose-Activated Switch Regulating Insulin Analog Secretion Enables Long-term Precise Glucose Control in Mice With Type 1 Diabetes.

Genetic modification of non-ß-cells to produce insulin is a promising therapeutic strategy for type 1 diabetes; however, it is associated with issues, including biosafety and precise regulation of insulin supply. In this study, a glucose-activated single-strand insulin analog (SIA) switch (GAIS) was constructed to achieve repeatable pulse activation of SIA secretion in response to hyperglycemia. In the GAIS system, the conditional aggregation domain-furin cleavage sequence-SIA fusion protein was encoded by the intramuscularly delivered plasmid and temporarily kept in the endoplasmic reticulum (ER) because it binds to the GRP78 protein; then, upon hyperglycemia, the SIA was released and secreted into the blood. In vitro and in vivo experiments systematically demonstrated the effects of the GAIS system, including glucose-activated and repeatable SIA secretion, long-term precise blood glucose control, recovered HbA1c levels, improved glucose tolerance, and ameliorated oxidative stress. Additionally, this system offers sufficient biosafety, as evidenced by the assays of immunological and inflammatory safety, ER stress, and histological evaluation. Compared with the viral delivery/expression system, the ex vivo implantation of engineered cells, and the exogenous inducer system, the GAIS system combines the advantages of biosafety, effectiveness, persistence, precision, and convenience, providing therapeutic potential for the treatment of type 1 diabetes. ARTICLE HIGHLIGHTS: We undertook this study to establish a glucose-responsive single-strand insulin analog (SIA) self-supply system in vivo. We sought to determine whether the endoplasmic reticulum (ER) can serve as a safe and temporary repository to store designed fusion proteins and release SIAs under hyperglycemic conditions for efficient blood glucose regulation. The intramuscularly expressed plasmid-encoded conditional aggregation domain-furin cleavage sequence-SIA fusion protein can be temporarily stored in the ER, and the SIA can be released under the stimulation of hyperglycemia, resulting in efficient and long-term regulation of stable blood glucose in mice with type 1 diabetes (T1D). The glucose-activated SIA switch system provides applicable potential for T1D therapy, integrating regulation and monitoring of blood glucose levels.

KIF4A knockdown inhibits tumor progression and promotes chemo-sensitivity via induction of P21 in lung cancer cells.

KIF4A has been demonstrated to play a crucial function in the pathogenesis of a broad number of tumors and have close association with PI3K/AKT pathway. The aim of this study was to explore the potential function of KIF4A in lung cancer progression by targeting PI3K/AKT pathway and P21 combination with doxorubicin. A549 cell lines were transfected with siRNA against KIF4A and negative control siRNA (si-NC). MTT assay and trypan blue staining were used to evaluate the effect of si-KIF4A on the doxorubicin cytotoxicity. The mRNA and protein expression levels of KIF4A and p21 were assessed by qRT-PCR and Western blotting. Apoptosis was measured by cell death ELISA kit. Our result revealed that KIF4A silencing decreased cellular proliferation in A549 lung cancer cells. Doxorubicin in combination with si-KIF4A led to significant reduction in the survival rate of A549 cell. KIF4A silencing upregulated p21. In conclusion, our results demonstrate that KIF4A inhibition sensitizes A549 cells to doxorubicin by targeting p21 and PI3K/AKT pathway, indicating a significant role for KIF4A in lung cancer chemotherapy.

18F-FDG-PET/CT-guided intensity-modulated radiotherapy for 42 FIGO III/IV ovarian cancer: A retrospective study.

The aim of the present study was to evaluate the curative effect of fludeoxyglucose-positron emission tomography/computed tomography (18F-FDG-PET/CT)-guided intensity-modulated radiotherapy (IMRT) for 42 patients with International Federation of Gynecology and Obstetrics (FIGO) stage III/IV ovarian cancer. Between January 2012 and December 2015, 42 patients with FIGO stage III/IV ovarian cancer who were treated with 18F-FDG-PET/CT-guided IMRT at the Department of Radiation Oncology were analyzed. A total of 21 patients who exhibited recurrence following surgery and 11 patients who were unable to tolerate or rejected surgery received 5-10 cycles of chemotherapy only. A total of 10 patients, who were either older (>70 years) or in poor general health were unable to undergo surgery and only received IMRT. The patients received a total radiation dose of 5,040 cGy (range, 4,500-5,500 cGy), with a dose fraction of 200 cGy/fx, administered a total of 10-14 times, 5 times/week, prior to being rested for half an hour to relocate lesions and undergoing a second round of radiotherapy for 10-14 cycles. The 1-, 2- and 3-year progression-free survival (PFS) rates of the patients were 66.7, 33.3 and 21.4%, respectively, and the median PFS time was 20.3 months. The 1-, 2- and 3-year local control rates of the patients were 90.5, 83.3 and 69.0%, respectively, and the 1-, 2- and 3-year overall survival (OS) rates were 73.8, 64.3 and 52.4%, respectively. According to the results of multivariate analysis using the Cox proportional hazards model, the Karnofsky performance status (KPS) score (1) was the only index associated with prognosis (P<0.003). The study concluded that for patients with advanced ovarian cancer, particularly for patients unable to undergo surgery or chemotherapy, 18F-FDG PET/CT-guided IMRT is a safe and effective treatment method, and it may be considered as an equally effective treatment option. Furthermore, the results of the present study suggested that the KPS score of a patient is the only factor affecting the OS time.

Curative effect of stereotactic body radiotherapy for unresectable massive primary liver cancer.

The aim of this study was to evaluate the curative effect of gamma knife stereotactic body radiotherapy (SBRT) for unresectable massive primary liver cancer. A total of 69 patients with unresectable massive (>10 cm) primary liver cancer who were treated by SBRT at the Department of Radiation Oncology of the 323 Hospital of People's Liberation Army (Xi'an, China) between October, 2006 and October, 2010, were analyzed. According to the Union for International Cancer Control TNM staging guidelines, the patients were graded as stage T1 (n=8), T2 (n=12), T3 (n=21) and T4 (n=28). None of the patients had lymph node metastasis, whereas 45 patients had portal vein tumor thrombosis. The Child-Pugh class was A (n=49), B (n=15) and C (n=5). The visible tumor volume ± standard deviation was 810±213 cm3. The patients received a total radiation dose of 50-60 Gy, with a dose fractionation of 4-6 Gy/fx, administered for a total of 9-12 times, 2-5 times/week. A total of 8 patients succumbed to the disease within 3 months after gamma knife treatment and were not included in the evaluation of the curative effect. The total effectiveness rate was 59.0% (36/61) and the median survival was 17.4 months for all the patients included in the study. The 1-, 2-, and 3-year overall survival rates were 71, 30 and 22%, respectively. In conclusion, SBRT appears to be effective for unresectable massive primary liver cancer.

Curative effect of stereotactic body radiotherapy on hepatic hilar carcinoma.

This study was conducted to investigate the effect of stereotactic body radiotherapy (SBRT) on hepatic hilar tumors. Between October, 2006 and October, 2012, we analyzed 63 unresectable hepatic hilar tumors that were treated by SBRT at the Department of Radiation Oncology, 323 Hospital of the People's Liberation Army, Xi'an, China. The patients received a total radiation dose of 45 Gy (range, 44-48 Gy) with a dose fractionation of 3-6 Gy/fx, administered for a total of 9-12 times, 2-5 times/week. At 1 and 3 months we evaluated therapeutic efficacy and 1- and 2-year survival rate. At 1 month, the patients exhibiting complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD) were 15 (23.8%), 34 (54.0%), 11 (17.5%) and 3 (4.7%), respectively. At 3 months, the number of cases with CR, PR, SD and PD was 22 (34.9%), 32 (50.8%), 3 (4.8%) and 6 (9.5%), respectively. The total effective rate, defined as CR + PR, was 85.7% (54/63). The number of patients with a tumor diameter of ≤5 cm in the CR, PR, SD and PD groups was 13 (72.2%), 4 (22.2%), 1 (5.6%) and 0 (0.0%), respectively. The number of patients with a tumor diameter of >5 cm in the CR, PR, SD and PD groups was 9 (20.0%), 28 (62.2%), 6 (13.3%) and 2 (4.5%), respectively. The 1-year survival rate of patients with a tumor diameter >5 cm was 71.4% (45/63) and the 2-year survival rate was 42.9% (27/63). In conclusion, SBRT appears to be a safe and effective treatment for hepatic hilar tumors.