The Establishment of Quantitatively Regulating Expression Cassette with sgRNA Targeting BIRC5 to Elucidate the Synergistic Pathway of Survivin with P-Glycoprotein in Cancer Multi-Drug Resistance

Quantitative analysis and regulating gene expression in cancer cells is an innovative method to study key genes in tumors, which conduces to analyze the biological function of the specific gene. In this study, we found the expression levels of Survivin protein (BIRC5) and P-glycoprotein (MDR1) in MCF-7/doxorubicin (DOX) cells (drug-resistant cells) were significantly higher than MCF-7 cells (wild-type cells). In order to explore the specific functions of BIRC5 gene in multi-drug resistance (MDR), a CRISPR/Cas9-mediated knocking-in tetracycline (Tet)-off regulatory system cell line was established, which enabled us to regulate the expression levels of Survivin quantitatively (clone 8 named MCF-7/Survivin was selected for further studies). Subsequently, the determination results of doxycycline-induced DOX efflux in MCF-7/Survivin cells implied that Survivin expression level was opposite to DOX accumulation in the cells. For example, when Survivin expression was down-regulated, DOX accumulation inside the MCF-7/Survivin cells was up-regulated, inducing strong apoptosis of cells (reversal index 118.07) by weakening the release of intracellular drug from MCF-7/Survivin cells. Also, down-regulation of Survivin resulted in reduced phosphorylation of PI3K, Akt, and mTOR in MCF-7/Survivin cells and significantly decreased P-gp expression. Previous studies had shown that PI3K/Akt/mTOR could regulate P-gp expression. Therefore, we speculated that Survivin might affect the expression of P-gp through PI3K/Akt/mTOR pathway. In summary, this quantitative method is not only valuable for studying the gene itself, but also can better analyze the biological phenomena related to it.

Matrine Reverses Multidrug Resistance in Eca-109/VCR Cells Via Inhibiting PI3K/Akt/mTOR Signaling Pathway

Objective To investigate the effect of matrine on multidrug resistance of Eca-100/VCR cells. Methods Methyl thiazolyl tetrazolium assay was used to assess the cytotoxic activity of matrine to Eca-109/VCR cells and the sensitivity of cells to Vincristine (VCR). Then Eca-109/VCR cells were divided into four groups: control group, matrine group, VCR group and matrine combined with VCR group. Immunofluorescence staining was used to detect P-gp protein expression. Western blot was used to detect MRP1, P-gp, Beclin 1, p62(SQSTM1), LC3, p-mTOR, p-Akt and p-p70S6K protein expression. MDR1 mRNA expression was detected by RT-qPCR. Transmission electron microscopy was used to detect the ultra structural changes. Results Matrine could reverse the resistance of Eca-109/VCR cells to VCR with a reversal index of 3.52. Compared with the other groups, the expression of LC3-II, Beclin 1 increased while P-gp, MRP1, p62(SQSTM1), p-mTOR, p-Akt and p-p70S6K significantly decreased in matrine+VCR group (P<0.05). The expression of MDR1 mRNA decreased in Eca-109/VCR cells treated with matrine, VCR or their combination. The number of autophagic vacuoles in Eca-109/VCR cells increased after matrine treatment. Conclusion Matrine has anticancer and anti multidrug resistance functions in Eca-100/VCR cells, and it may be produced by promoting autophagy which mediated through PI3K/Akt/mTOR signaling pathway.

Discovering and Characterizing of Survivin Dominant Negative Mutants With Stronger Pro-apoptotic Activity on Cancer Cells and CSCs

Survivin as a member of the inhibitor of apoptosis proteins (IAPs) family is undetectable in normal cells, but highly expressed in cancer cells and cancer stem cells (CSCs) which makes it an attractive target in cancer therapy. Survivin dominant negative mutants have been reported as competitive inhibitors of endogenous survivin protein in cancer cells. However, there is a lack of systematic comparative studies on which mutants have stronger effect on promoting apoptosis in cancer cells, which will hinder the development of novel anti-cancer drugs. Here, based on the previous study of survivin and its analysis of the relationship between structure and function, we designed and constructed a series of different amino acid mutants from survivin (TmSm34, TmSm48, TmSm84, TmSm34/48, TmSm34/84, and TmSm34/48/84) fused cell-permeable peptide TATm at the N-terminus, and a dominant negative mutant TmSm34/84 with stronger pro-apoptotic activity was selected and evaluated systematically in vitro. The double-site mutant of survivin (TmSm34/84) showed more robust pro-apoptotic activity against A549 cells than others, and could reverse the resistance of A549 CSCs to adriamycin (ADM) (reversal index up to 7.01) by decreasing the expression levels of survivin, P-gp, and Bcl-2 while increasing cleaved caspase-3 in CSCs. This study indicated the selected survivin dominant negative mutant TmSm34/84 is promising to be an excellent candidate for recombinant anti-cancer protein by promoting apoptosis of cancer cells and their stem cells and sensitizing chemotherapeutic drugs.

Compound Injection of Shenqi as a Reversal Drug on Docetaxel Resistant Human Lung Adenocarcinoma Cell A549/DTX

To investigate the effect and the mechanism of compound injection of Shenqi (Codonopsis and Astragalus) on Docetaxel resistance of human lung adenocarcinoma cell A549/ DTX. The cytotoxicities of Shenqi Injection were assayed with the MTT method, the effects of Shenqi Injection on apoptosis induced by Docetaxel in A549/ DTX was examined by flow cytometry (FCM). The expression levels of apoptosis regulator Bc1-2 and Bax were detected by Western blot. After treatment with Shenqi Injection for 24 hours, results showed that 15 μL / mL Shenqi Injection significantly increased toxicity of Docetaxel on A549/DTX. Shenqi Injection could reverse the Docetaxel resistance in A549/ DTX in vitro. The drug resistance reversal index (RI) was equal to 2.71 analyzed with MTT method. Shenqi Injection could enhance the apoptosis effect of Docetaxel on A549/DTX. We conclude that Shenqi Injection can reverse the drug resistance on A549/DTX, and its mechanism is to promote apoptosis may be related with the up-regulation of Bc1-2 expression and down-regulation of Bax expression.