Inhibition Of Epothilone-bacterial Ghosts Systems On Proliferation Of Tumor Cells

The epothilones, a new class of microtubule targeting agents seem to be a very promising alternative to the current strategy of cancer treatment. However, their use are limited by the toxic side-effects and the poor aqueous solubility. To address this challenge, we explored a new strategy of Epothilone B (EpoB) delivery, using bacterial ghosts (BGs) from E.coli as drug delivery vector. Bacterial ghosts are non-denatured envelopes of Gram-negative bacteria with fully intact surface structures for specific attachment to mammalian cells.Bacterial ghosts are produced by the controlled expression of bacteriophage PhiX174 lysis gene, in order to produce bacterial ghost, the lysis plasmid pBV220-geneE were constructed. The plasmid was successfully transformed into the strain BL21(DE3). Generation of BL21(DE3) ghosts was performed by shifting the incubation temperature to 42℃ to inactivate the repressor protein and activate lysis geneE. Onset of the lysis occurred 30 min after temperature elevation because the number of viable cells began to decrease after induction, and the lysis process was completed 2 h after induction. The results of three replicate experiments showed the efficiency of BL21(DE3) induction was 99.99% at the end of the lysis process. Electron microscopic analysis of BL21(DE3) revealed no gross alterations in cellular morphology compared to unlysed cells except for the lysis pore. Pores ranging from 40 to 200 nm in diameter were observed by scanning electron microscopy. The structural integrity and the loss of cytoplasmic materials were observed in BL21(DE3) by transmission electron microscopy.Treated the logarithmic phase Hela cells with different kinds of drugs. The cytotoxic activity of BGs-EpoB and naked EpoB were determined by the MTT test. The results obtained demonstrated that the antiproliferative capacity of BGs-EpoB in Hela cell line (measured as IC50 after 48 h continuous treatment) was two times greater than that of naked EpoB’s. After BGs-EpoB and EpoB treated, apoptotic Hela cell levels were measured by double staining with propidium iodide (PI) as well as Annexin V staining. Through the transmission electron microscopy (TEM) observe, we found that the BGs-EpoB induced more changes in morphology of Hela cell than naked EpoB at the same dose. The results provide direct evidence that BGs-EpoB is considering more cytotoxic to Hela cells than EpoB at the same dose.Then, we study the effects of EpoB and BG-EpoB on EpCAM expression of Hela cells at different conditions. We use the Real time fluorogenic quantitative PCR (RTFQ PCR) methods to analyze the amount of EpCAM expression, use the GAPDH as reference gene. The results indicate that both of the two forms of EpoB can makes the EpCAM mRNA expression a little lower, but there is no significance level between them. We should look for more sensitive index to show the advantage of BG-EpoB.