Synergistic antitumor activity of vitamin D3 combined with metformin in human breast carcinoma MDA-MB-231 cells involves m-TOR related signaling pathways.

Metformin is usually used for the treatment of type 2 diabetes. Recently, many studies suggest that metformin and vitamin D have broad-spectrum antitumor activities. Our aim in this research was to study the effects of vitamin D3 combined with metformin on the apoptosis induction and its mechanisms in the human breast cancer cell line MDA-MB-231. Cell proliferation was measured by methylthiazol tetrazolium (MTT) assay. The morphology of cell apoptosis was observed after Hoechst 33342 staining. Here we show that vitamin D3 280 µg/ml or vitamin D3 300 µg/ml or vitamin D3 320 µg/ml seperately combined with metformin 15000 µg/ml exhibited synergistic effects on cell proliferation and apoptosis. The underlying anti-tumor mechanisms may involve m-TOR related pathways, which are related to activating expression of cleaved caspase-3, Bax and p-AMPK, as well as inhibiting expressions of p-Bcl-2, c-Myc, p-IGF-IR, p-mTOR, p-P70S6K, p-S6.

Vitamin D3 enhances antitumor activity of metformin in human bladder carcinoma SW-780 cells.

OBJECTIVE: To study the effects of vitamin D3 combined with metformin on the proliferation and apoptosis in human bladder cancer cell line SW-780 and its possible mechanism. METHODS: MTT assay and fluorescence microscope observations were used to study the effects of vitamin D3 combined with metformin on the proliferation and apoptosis of SW-780 cells in vitro. Western blot was used to detect the expression of apoptosis-related proteins p-Bcl-2, Bax, Cyclin D1, c-Myc and related signaling pathways activated proteins p-IGF-IR, p-mTOR, p-P70S6K, p-S6. RESULTS: MTT results showed that 320 µg/ml vitamin D3 combined with 620 µg/ml metformin acting on cells for 48h had a significant synergistic effect on proliferation. Fluorescence microscope observations showed that compared with negative control group and monotherapy treatment group, the apoptosis features of combination treatment group were obvious and the apoptosis rate increased greatly. Western blot showed that compared with the negative control group and monotherapy treatment group, the expression levels of p-Bcl-2, Cyclin D1 and c-Myc in combination treatment group significantly decreased, whereas the expression level of Bax significantly increased, and the expression levels of p-IGF-IR, p-mTOR, p-P70S6K and p-S6 in combination treatment group significantly decreased. CONCLUSION: Vitamin D3 combined with metformin exhibited obvious inhibitory effects on the cell proliferation and apoptosis induction in SW-780 cells. The underlying anti-tumor mechanism might be related to inhibiting the expressions of p-Bcl-2, Cyclin D1, c-Myc, p-IGF-IR, p-mTOR, p-P70S6K, p-S6 and activating the expression of Bax.

Therapeutic effect of ginsenoside Rd in rats with TNBS-induced recurrent ulcerative colitis.

Ulcerative colitis (UC) is characterized by oxidative and nitrosative stress and neutrophil infiltration. In the present study, we aimed to investigate the therapeutic effect of ginsenoside Rd (GRd) in rats with 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced recurrent UC. After UC was twice-induced by intracolonic injection of TNBS, rats were intragastrically administered different doses of GRd per day for 7 days. The colonic lesions and inflammation were evaluated both histologically and biochemically. Compared with the TNBS group, GRd treatment facilitated recovery of pathologic changes in the colon after induction of recurrent UC, as evidenced by a significant reduction of colonic weight/length ratio and macroscopic and microscopic damage scores (p < 0.01). The myeloperoxidase and inducible nitric oxide synthase activities with malonyldialdehyde and nitric oxide levels in colonic tissues were significantly decreased in the GRd group compared with those in the TNBS group (p < 0.01). GRd treatment was associated with remarkably increased superoxide dismutase and glutathione peroxidase activities. Results showed a valuable effect of GRd against TNBS-induced recurrent UC by inhibiting neutrophil infiltration and promoting the antioxidant capacity of the damaged colonic tissue.

Therapeutic effects of a recombinant mutant of the human ciliary neurotrophic factor in a mouse model of metabolic syndrome.

Metabolic syndrome (MS) is highly prevalent in developed countries and becoming a serious worldwide public health issue. In this study, we established a MS model by feeding male C57BL/6J mice with a high-fat diet (10%) for 18.5 weeks, studied the therapeutic effects of a recombinant mutant of the human ciliary neurotrophic factor (rhmCNTF) 0.1 (C-0.1) or 0.3 (C-0.3) mg x kg(-1) per day subcutaneously or pair feeding (PF, which mice were restricted to the same amount of food as eaten by C-0.3 treated mice) in MS mice. After 10 days treatment, rhmCNTF reduced obesity related indices, ameliorated glucose and lipid metabolism abnormality, and enhanced insulin sensitivity. In addition, liver function and antioxidant ability of MS mice were improved by rhmCNTF. Pair feeding revealed the same effects as C-0.3 on obesity related indices and insulin sensitivity, but aggravated hepatic steatosis and hepatic function. The results suggest that rhmCNTF could serve as an effective therapeutic agent for MS and related diseases.

The relationship between structure and antioxidative activity of piperidine nitroxides.

We have investigated the relationship between structure and antioxidative activity of piperidine nitroxides which were substituted by different groups at the 4-position. All of the tested piperidine nitroxides inhibited malondialdehyde (MDA) generation caused either spontaneously or by a hydroxyl free radical generation system (Fe2+-ascorbic acid) in homogenates of liver, heart and kidney of rats, and antagonized H2O2-induced haemolysis from rat erythrocytes in a concentration-dependent manner. The same rank was followed: Bis-(4-amino-2,2,6,6-tetramethyl piperidinooxyl) (4-BIS-Tempo) and 4-azido-2,2,6,6-tetramethyl piperidinooxyl (4-N(3)-Tempo) > 4-isothiocyanate-2,2,6,6-tetramethyl piperidinooxyl (4-ISO-Tempo), 4-2', 4'-dinitrophenylhy-drazone-2,2,6,6-tetramethyl piperidinooxyl (4-D-Tempo), 4-sulfonate-2,2,6,6-tetramethyl piperidinooxyl (4-S-Tempo) and 4-amino-2,2,6,6-tetramethyl piperidinooxyl (4-NH(2)-Tempo) > 4-acetate ester-2,2,6,6-tetramethyl piperidinooxyl (4-A-Tempo) and 4-benzoate-2,2,6,6-tetramethyl piperidinooxyl (4-B-Tempo). With the exception of 4-A-Tempo and 4-D-Tempo, the tested piperidine nitroxides inhibited superoxide anion (O(2*-)) release from neutrophils stimulated by zymosan. The concentration required for inhibiting O(2*-) release was higher than that of inhibiting MDA formation and haemolysis. However, 4-amino-2,2,6,6-tetramethyl piperidine (4-NH2-TempH) and other 4-position substitutes, such as NaN3 and isothiocyanate, had no effects on MDA formation, haemolysis or O(2*-) release. The results indicated that nitroxides have a wide range of scavenging reactive oxygen species (ROS) actions. The nitroxide moiety was the essential group while the 4-position substitutes could influence the activity of nitroxides on scavenging ROS.

Amelioration of doxorubicin-induced myocardial oxidative stress and immunosuppression by grape seed proanthocyanidins in tumour-bearing mice.

We have investigated the protective effects of grape seed proanthocyanidins on doxorubicin-induced toxicity in tumour-bearing mice. The intraperitoneal administration of doxorubicin (2 mg kg(-1) every other day, cumulative dosage for 18 mg kg(-1)) significantly inhibited the growth of sarcoma 180, and induced myocardial oxidative stress with decreased superoxide dismutase and glutathione peroxidase activity while increasing malondialdehyde formation in the heart or serum. Doxorubicin-induced myocardial oxidative stress also reduced lactate dehydrogenase and creatine kinase activity in the heart and elevated their levels in the serum. Doxorubicin also affected immune functions of tumour-bearing mice with significantly decreased interleukin-2 (IL-2) and interferon-gamma (INF-gamma) production, and slightly decreased natural killer (NK) cell cytotoxicity, lymphocyte proliferation and CD4+/CD8+ ratio. It markedly increased the percentages of cytotoxic T cells (CD3+CD8+), helper T cells (CD3+CD4+), IL-2R+CD4+, and IL-2R+ cells as compared with untreated tumour-bearing mice. The intragastric administration of proanthocyanidin (200 mg kg(-1) daily) significantly inhibited tumour growth, and increased NK cell cytotoxicity, lymphocyte proliferation, CD4+/CD8+ ratio, IL-2 and INF-gamma production. Moreover, proanthocyanidin strongly enhanced the anti-tumour effect of doxorubicin and the above immune responses, and completely eliminated myocardial oxidative stress induced by doxorubicin. In conclusion, intragastric administration of proanthocyanidin could enhance the anti-tumour activity of doxorubicin and ameliorate doxorubicin-induced myocardial oxidative stress and immunosuppression in tumour-bearing mice.

Protective effect of 1-(2,6-dimethylphenoxy)-2-(3,4-dimethoxyphenylethylamino) propane hydrochloride on myocardial ischemia-reperfusion injury in rats.

The aim of the present study was to investigate the protective effect of 1-(2,6-dimethylphenoxy)-2-(3,4-dimethoxyphenylethylamino) propane hydrochloride (DDPH) on myocardial ischemia-reperfusion (I/R) injury in rats and the mechanism of its myocardial protection. For this purpose, 50 Wistar rats were divided into five groups: sham group, control group, verapamil treated group, and two DDPH treated groups (20 and 40 mg/kg, respectively). Myocardial I/R injury model was established by reperfusion for 120 min after 40 min ischemia induced by the ligation of left descending coronary artery in rats. The influence of DDPH on myocardial infarction size was observed and the levels of myocardial enzymes in serum were measured. The activities of oxygen free radical scavenging enzymes and the content of malondialdehyde (MDA) in myocardium and serum were determined. The pathological changes of myocardial tissue were observed. The results showed that DDPH significantly diminished myocardial infarction size, reduced the release of myocardial creatine phosphokinase (CPK), lactate dehydrogenase (LDH) and glutamic oxaloacetic aminotransferase (GOT), protected the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and decreased the content of MDA in myocardium and serum as compared with the control group. The degree of myocardial injury was slighter in DDPH treated groups than in control group. These results suggest that DDPH produces a cardioprotective effect during myocardial I/R injury, which may be related to blocking calcium channels and inhibiting the formation of the oxygen free radical and subsequent peroxidation of lipid by DDPH.

Protective effects of pentadecapeptide BPC 157 on gastric ulcer in rats.

AIM: To investigate the protective effects of gastric pentadecapeptide BPC 157 on acute and chronic gastric ulcers in rats and to compare the results in therapy of human gastric ulcers by different administration methods. METHODS: Gastric pentadecapeptide BPC 157 was administered (initial single or continuous administration) into rats either intragastrically or intramuscularly before (induced acute gastric ulcer) or after (induced chronic gastric ulcer) the applications of inducing agents, and each animal was sacrificed to observe the protective effects of BPC 157 on gastric ulcers. RESULTS: Both intramuscular (im) and intragastric (ig) administration of BPC 157 could apparently reduce the ulcer area and accelerate the healing of induced ulcer in different models and the effect of im administered BPC 157 was better than that of ig. The rats treated with higher dosages (400 ng/kg, 800 ng/kg) of BPC 157 (im and ig) showed significantly less lesion (P<0.01 vs excipient or saline control), the inhibition ratio of ulcer formation varied between 45.7% and 65.6%, from all measurements except 400 ng/kg BPC 157 in pylorus ligation induced model (P<0.05), in which the inhibition rate was 54.2%. When im administered (800 ng/kg BPC 157) in three models, the inhibition ratio of ulcer formation was 65.5%, 65.6% and 59.9%, respectively, which was better than that of famotidine (its inhibition rate was 60.8%, 57.2% and 34.3%, respectively). Continuous application of BPC 157 (in chronic acetate induced gastric ulcer) could accelerate rebuilding of glandular epithelium and formation of granulation tissue (P<0.05 at 200 ng/kg and P<0.01 at 400 ng/kg and 800 ng/kg vs excipient or saline control). CONCLUSION: Both im and ig administered gastric pentadecapeptide BPC 157 can apparently ameliorate acute gastric ulcer in rats and antagonize the protracted effect of acetate challenge on chronic ulcer. The effect of im administration of BPC 157 is better than that of ig, and the effective dosage of the former is lower than that of the latter.

[Effect of subcutaneous implant of peripheral nerve allograft on sciatic nerve regeneration in rats].

OBJECTIVE: To study the effect of subcutaneous implant of peripheral nerve allograft on sciatic nerve regeneration in rats. METHODS: Out of 30 male Wistar rats, 6 were donors and 24 were divided randomly into 2 groups. In experimental group (group A, n = 12), a 15 mm segment of sciatic nerve harvested from donors was separately inserted into subcutaneous compartment on the right thigh; two weeks later, the segment of sciatic nerve in subcutaneous compartment was removed and transplanted into a 10 mm sciatic nerve defect of left, which was made immediately. In the control group (group B, n = 12), a 10 mm sciatic nerve defect was made and immediately repaired in situ on the left thigh. The regeneration of sciatic nerve was examined histologically (after 2, 4, 8, and 14 weeks) and electrophysiologically (after 14 weeks of operation). RESULTS: After 2 weeks of operation, the inflammatory reaction was a little stronger in group A than in group B. After 4 weeks, the intensity of the inflammatory reaction was similar between two groups; some collagen fibers proliferated. After 8 weeks, the inflammatory reaction ended and the collagen fibers proliferated obviously. After 14 weeks of operation, the structure of epineurium was in integrity and there was no obvious difference in perineurium and endonurium between two groups. A large number of myelinated nerve fibers and a small number of unmyelinated nerve fibers regenerated. The structure of myelin sheath was in integrity. The number and size of regenerated axon had no significant difference between two groups(P > 0.05). The conduction velocity, the peak value and the latent period of motor nerve were no significant difference between two groups (P > 0.05). CONCLUSION: The allograft of sciatic nerve inserted into subcutaneous compartment can promote nerve regeneration.