KLF4 functions as an oncogene in promoting cancer stem cell-like characteristics in osteosarcoma cells.

Despite more effective chemotherapy combined with limb-salvage surgery for the osteosarcoma treatment, survival rates for osteosarcoma patients have stagnated over the past three decades due to the poor prognosis. Osteosarcoma cancer stem cells (OSCs) are responsible for the growth and metastasis of osteosarcoma. The existence of OSCs offers a theoretical explanation for therapeutic failures including tumor recurrence, metastasis, and drug resistance. Understanding the pathways that regulate properties of OSCs may shed light on mechanisms that lead to osteosarcoma and suggest better modes of treatment. In this study, we showed that the expression level of Kruppel-like factor 4 (KLF4) is highly associated with human osteosarcoma cancer stemness. KLF4-overexpressed osteosarcoma cells displayed characteristics of OSCs: increased sphere-forming potential, enhanced levels of stemness-associated genes, great chemoresistance to adriamycin and CDDP, as well as more metastasis potential. Inversely, KLF4 knockdown could reduce colony formation in vitro and inhibit tumorigenesis in vivo, supporting an oncogenic role for KLF4 in osteosarcoma pathogenesis. Furthermore, KLF4 was shown to activate the p38 MAPK signaling pathway to promote cancer stemness. Altogether, our studies uncover an essential role for KLF4 in regulation of OSCs and identify KLF4-p38 MAPK axis as a potential therapeutic target for osteosarcoma treatment.

Nuclear translocation and activation of YAP by hypoxia contributes to the chemoresistance of SN38 in hepatocellular carcinoma cells.

Although hypoxia is a prominent feature contributing to the therapeutic resistance of hepatocellular carcinoma cells (HCC) against chemotherapeutic agents, including the Topoisomerase I inhibitor SN38, the underlying mechanism is not fully understood and its understanding remains a major clinical challenge. In the present study, we found that hypoxia-induced nuclear translocation and accumulation of YAP acted as a survival input to promote resistance to SN38 in HCC. The induction of YAP by hypoxia was not mediated by HIF-1α because manipulating the abundance of HIF-1α with CoCl2, exogenous expression, and RNA interference had no effect on the phosphorylation or total levels of YAP. The mevalonate-HMG-CoA reductase (HMGCR) pathway may modulate the YAP activation under hypoxia. Combined YAP inhibition using either siRNA or the HMGCR inhibitor statins together with SN38 treatment produced improved anti-cancer effects in HCC cells. The increased anti-cancer effect of the combined treatment with statins and irinotecan (the prodrug of SN-38) was further validated in a human HepG2 xenograft model of HCC in nude mice. Taken together, our findings identify YAP as a novel mediator of hypoxic-resistance to SN38. These results suggest that the administration of SN28 together with the suppression of YAP using statins is a promising strategy for enhancing the treatment response in HCC patients, particularly in advanced stage HCC cases presenting hypoxic resistance.

[Construction of expression vector pLCK-CD69-IRES-EGFP and generation of CD69 transgenic mice].

OBJECTIVE: To construct the expression vector pLCK-CD69-IRES-EGFP that contains mouse cell surface activation protein CD69 and enhanced green fluorescent protein(EGFP),and to generate CD69 transgenic mice based on this vector. METHODS: First, RNA was extracted from mouse lung tissue and cDNA was synthesized via reverse transcription. PCR primer was designed through the PubMed searching, then mouse CD69 DNA fragment was amplified with PCR. Second, this DNA fragment was subcloned to the pInsulater-LCK-IRES-EGFP plasmid and constructed the transgenic vector after the verification of nucleotide sequence. Third, the expression vector was then transfected into 293 T cells and its expression in 293 T cells was observed under fluorescence microscope. Last, microinjection was performed to transfer the expression vector pLCK-CD69-IRES-EGFP into fertilized eggs, which were implanted into pseudo-pregnant recipient mice. After birth the tail samples of the pups were obtained for the purpose of genotyping to determine the transgenic founders. Fluorescence microscope and flow cytometer were used to measure the expression of CD69 on cells. RESULTS: The construction of the expression vector pLCK-CD69-IRES-EGFP was verified by enzyme digestion and DNA sequencing. The transfected 293 T cell showed expression of the protein under fluorescence microscope. Identification of PCR for the tail tissue of the pups confirmed the present of CD69 transgene and resting lymphocytes demonstrated the expression of CD69. CONCLUSION: The construction of expression vector pLCK-CD69-IRES-EGFP and generation of CD69 transgenic mice have been successfully processed, which lays a foundation of the solid pattern studies in inflammatory diseases.

Antimetastatic activity of MONCPT in preclinical melanoma mice model.

Previous study demonstrated that MONCPT, a topoisomerase I inhibitor, exhibited potent anti-proliferation and anti-angiogenesis activity in vitro and in vivo. In this study, we report the efficacy of MONCPT against the development of melanoma metastasis by an intravenous injection of green fluorescent protein-transfected mice melanoma carcinoma (B16F10-GFP) cells in C57BL/6 mice. MONCPT (2.0, 5.0 and 12.5 mg/kg/2 days) markedly decreased B16F10-GFP pulmonary metastases by 12.8%, 53.1% and 76.3%, respectively; whereas higher doses of MONCPT (31.0 mg/kg/2 days) significantly inhibited the tumor growth of B16F10 xenograft model. In the in vitro experiment, MONCPT suppressed the B16F10-GFP cell invasion and migration without affecting cell survival. Further studies demonstrated that MONCPT decreased the secretion of matrix metalloproteinase (MMP)-9 and VEGF, and reduced the protein expression of HIF-1α as well as the phosphorylation level of ERK in B16F10-GFP cells. These in vivo and in vitro results indicate that MONCPT possesses both the potent antimetastatic ability and the tumor growth-inhibition activity, and the dual function promises MONCPT as a potential therapeutic agent for tumor metastasis and tumor growth of melanoma carcinoma.

Insulin sensitizing activity of ethyl acetate fraction of Acorus calamus L. in vitro and in vivo.

UNLABELLED: Acorus calamus L. (AC), family Araceae, have been used in the Indian and Chinese systems of medicine for hundreds of years. The radix of AC is widely used in the therapy of diabetes in traditional folk medicine of America and Indonesia. AIM OF THE STUDY: To investigate the insulin sensitizing activity and antidiabetic effects of the ethyl acetate fraction of AC (ACE). MATERIALS AND METHODS: Glucose consumption mediated by insulin was detected in L6 rat skeletal muscle cells. Diabetes and its complications related indexes were monitored after orally administrating to genetically obese diabetic C57BL/Ks db/db mice daily for 3 weeks. RESULTS: ACE (12.5 and 25 microg/ml) increased glucose consumption mediated by insulin in L6 cells (p<0.05 and p<0.01). In db/db mice, ACE (100 mg/kg) significantly reduced serum glucose, triglyceride, reinforce the decrease of total cholesterol caused by rosiglitazone (at least p<0.05), and markedly reduced free fatty acid (FFA) levels and increased adiponectin levels (p<0.01 and p<0.05) as rosiglitazone did (p<0.05 and p<0.001). Serum insulin was decreased but not significantly. In addition, ACE decreased the intake of food and water, and did not increase body weight gain whereas rosiglitazone did. CONCLUSIONS: Owing to the ability of insulin sensitizing, ACE has the potential to be useful for the treatment of diabetes and cardiovascular complications without body weight gain.

[Transport mechanism of danshensu across Caco-2 monolayer model].

OBJECTIVE: To study the absorption mechanism of Danshensu by using Caco-2 monolayer model . METHOD: Caco-2 cell monolayer model was used to study the bi-direction transport of Danshensu. An LC-MS method was developed to measure the concentration of Danshensu in cell culture medium and calculate the apparent permeability coefficients (P(app)). The effects of time, drug concentration and inhibitor on the absorption of Danshensu were studied. RESULT: Transport of Danshensu was time and concentration dependent, and it was also effected by P-glycoprotein inhibitor. P(app) increased with time increase and tended to become saturated at some point. It, however,decreased while concentration of Danshensu increased. P(ratio) is larger than 1.5 . Verapamil can cause significantly effect on transport of Danshensu: P(app,A-B) increased and P(app,B-A) decreased . CONCLUSION: The absorption of Danshensu in Caco-2 cell model may be an active transportation mediated by P-glycoprotein transporter.

[Effects of Gardenia-aweto compound by different extraction method on antagonizing acute respiratory distress syndrome].

OBJECTIVE: To study the effect of Gardenia-Aweto compound (GAC) and two component on preventing acute respiratory distress syndrome (ARDS) by the rabbit model of ARDS induced by intravenous injection of oleic acid. To detect the efficiency component of GAC in preventing ARDS. METHOD: GAC was divided into two compounts, ethanol-soluble components (ESC) and ethanol-deposition components (EDC), based on polarity. Forty-three new zealand rabbits were randomly divided into five groups, the blank control group, the model group, the GAC groups, the ESC group, and the EDC group. The ARDS model was induced by intravenous injection of oleic acid. Dynamic changes of arterial blood gas, lung index, albumin in bronchoalveolar lavage fluid (BALF) in different groups and lung histological changes were observed and compared. RESULT: As compared with the blank group, in the model group, GAC group, ESC group, EDC group the arterial PO2 and oxygen saturation deprived continuously. While SO2 in GAC group at time points 30, 60, 90, 120 min (P < 0.05 or 0.01) and SO2 in ESC group at time points 30, 60, 90 min were higher than those in ARDS group. PO2 in ESC group at time points 30, 60 min (P < 0.05) were higher than those in ARDS group. The value of LI and W/D were higher in ARDS group than in sham group (P < 0.01), they were much lower in HD group than in ARDS group (P < 0.01). Concentration of BALF-albumin increased markedly in ARDS group and pre-treatment groups compared with sham group, but it was much lower in GAC group and ESC group, there was a significant difference between GAC group (P < 0.01), ESC group (P < 0.05) and ARDS group. The lung histological changes had been improved in GAC group and ESC group. But no significantly difference between above-mentioned parameters was found in comparison in the model group and in the EDC group. CONCLUSION: Preventive administration of GAC or ESC an protect the damaged lung function in ARDS rabbits induced by oleic acid. The efficiency component of GAC in preventing ARDS is ESC. GAC antagonizing ARDS may relate to its anti-inflammatory, immuno-modulatory, anti-oxidant and antithrombotic effects.