Metformin reverses the drug resistance of cisplatin in irradiated CNE-1 human nasopharyngeal carcinoma cells through PECAM-1 mediated MRPs down-regulation.

Objective: To explore a way to reverse the drug resistance for irradiated CNE-1 human nasopharyngeal carcinoma cells and try to develop a new high efficacy with low toxicity therapeutic approach. Methods: 300 Gy irradiated the CNE-1 human nasopharyngeal carcinoma cells, and then treated with single-agent cisplatin or metformin, or combination of both drugs. MTT assay and FCM were applied to detect cell viability and apoptosis. Western blot and RT-PCR were used to characterize the protein and mRNA expression after various drug administrations. Results: The results presented single-agent metformin was capable of arresting the tumor growth and inducing apoptosis in irradiated CNE-1 cells and also demonstrated a synergy effect with cisplatin. Furthermore, metformin down-regulates the PECAM-1 expression, which could regulate Multi-drug Resistance-associate Proteins (MRPs) expression leading to cisplatin resistance of irradiated CNE-1 cells. A pan-MRP inhibitor, probenecid, can resecure cisplatin resistance leading by radiation. Conclusions: Metformin, due to its independent effects on PECAM-1, had a unique anti-proliferative effect on irradiated CNE-1 cells. It would be a new therapeutic option to conquer cisplatin resistance for advanced NPC patients after radiotherapy.

Construction and evaluation of luciferase reporter phages for the detection of active and non-replicating tubercle bacilli.

The luciferase reporter phages (LRP) show great promise for diagnostic mycobacteriology. Though conventional constructs developed from lytic phages such as D29 and TM4 are highly specific, they lack sensitivity. We have isolated and characterized Che12, the first true temperate phage infecting M. tuberculosis. Since the tuberculosis (TB) cases among HIV infected population result from the reactivation of latent bacilli, it would be useful to develop LRP that can detect dormant bacteria. During dormancy, pathogenic mycobacteria switch their metabolism involving divergent genes than during normal, active growth phase. Since the promoters of these genes can potentially function during dormancy, they were exploited for the construction of novel mycobacterial luciferase reporter phages. The promoters of hsp60, isocitrate lyase (icl), and alpha crystallin (acr) genes from M. tuberculosis were used for expressing firefly luciferase gene (FFlux) in both Che12 and TM4 phages and their efficiency was evaluated in detecting dormant bacteria from clinical isolates of M. tuberculosis. These LRP constructs exhibited detectable luciferase activity in dormant as well as in actively growing M. tuberculosis. The TM4 ts mutant based constructs showed about one log increase in light output in three of the five tested clinical isolates and in M. tuberculosis H37Rv compared to conventional lytic reporter phage, phAE129. By refining the LRP assay format further, an ideal rapid assay can be designed not only to diagnose active and dormant TB but also to differentiate the species and to find their drug susceptibility pattern.

Expression pattern of matrix metalloproteinases-13 in a rat model of alcoholic liver fibrosis.

BACKGROUND: Interstitial collagenase has been considered as an essential enzyme for collagenolysis in liver fibrosis, because type I and III collagens increase predominantly in liver fibrosis. The present study aimed to demonstrate the gene expression of matrix metalloproteinases-13 (MMP-13) in the progressive phases of ethanol induced experimental liver fibrosis in rats. METHODS: Thirty-four Sprague-Dawley rats were randomly divided into two groups. The experimental group (24 rats) was given ethanol (44%, 7 g/kg) every day and the control group (10) was given normal saline. Liver samples were harvested from experimental rats at 4, 12 and 24 weeks respectively. The kinetics of MMP-13 mRNA expression was assayed by semi-quantity reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: In normal rat liver, a faint band for MMP-13 mRNA was observed by RT-PCR (0.24+/-0.41). The gene expression of MMP-13 was increased in the liver of the rats treated with ethanol for 4 weeks (0.62+/-0.54), but it was not considered statistically significant (P>0.05). And the livers from 12-week-treated rats showed a marked mRNA expression (1.65+/-0.47, P<0.01). Once fibrosis became prominent (24 weeks), a faint band of MMP-13 mRNA was observed (0.39+/-0.25). CONCLUSION: MMP-13 participates in the degradation of newly-formed matrix in the early phase of rat liver fibrosis induced by ethanol, and it was induced in a distinct time frame.

[Gene expression of interstitial collagenase MMP-13 in progressive phase of rat liver fibrosis induced by ethanol].

OBJECTIVES: To demonstrate the gene expression of MMP-13 in the progressive phase of ethanol-induced experimental liver fibrosis in rats. METHODS: 34 SD rats were randomized into two groups. The rats in experimental group (n=24) were given ethanol (44%, 7g/kg) every day, and the rats in control group (n=10) were given equality normal saline. Liver samples were harvested from experimental rats at the 4th, 12th and 24th weeks respectively. The dynamic expression of MMP-13 mRNA was assayed by semi-quantity reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: In normal rat liver, a faint band of MMP-13 mRNA was observed by RT-PCR (0.24+/-0.41). The gene expression of MMP-13 increased in the livers of rats treated with ethanol for 4 weeks (0.62+/-0.54), but it was not considered statistically, when compared with that in normal rats livers. And the livers from 12-week-treated rats showed a markedly MMP-13 mRNA expression (1.65+/-0.47, t=-4.363, P<0.01). Once the fibrosis became prominent (24 weeks), a faint band of MMP-13 mRNA was observed (0.39+/-0.25). CONCLUSION: MMP-13 participates in the degradation of newly-formed matrix in the early phase of rat liver fibrosis induced by ethanol, but it expresses in a distinct time frame