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  • br In the following studies we have investigated the abiliti

    2020-03-24


    In the following studies, we have investigated the abilities of NAX compounds to interact with metformin and inhibit the growth of PDAC cell lines. We have investigated the effects of the introduction of one phenyl moiety (NAX012, NAX042), two phenyl moieties (benzhydryl) (NAX035, NAX053) and one heterocycle (pyridine) (NAX075, NAX077). In the monophenyl series, we in-vestigated also the effects of single or multiple electron-releasing (NAX038, NAX054) or electron-withdrawing (NAX014, NAX060, NAX111) substituents. The effects of two different lengths of the hydrocarbon linker were also examined. Some of the NAX com-pounds could interact with metformin and inhibit proliferation better than others. The presence of WT-TP53 was determined to influence the electiveness of certain NAX compounds upon co-treatment with metformin.
    2. Materials and methods
    2.1. Cell lines and tissue culture
    Cells were cultured in Dulbecco's modified Eagles medium, (DMEM), (Invitrogen, Carlsbad, CA), SPDP containing L-gluta-mine medium supplemented with 5% fetal bovine serum (FBS) purchased from Atlanta Biologicals, Atlanta, GA, USA) as described in
    S.M. Akula, et al. Advances in Biological Regulation xxx (xxxx) xxxx
    Fig. 5. Effects of Different Doses of Metformin in the Presence of No Drugs, 100 nM Berberine, 100 nM NAX038 or 100 nM NAX060 on Colony Formation in AsPC-1 and BxPC-3 PDAC Cells. The effects of either no BBR or 100 nM BBR on the colony formation in the presence of different doses of metformin (MET) in AsPC-1 + pLXSN (Adh.) (Panel A) and BxPC-3 + pLXSN (Adh) (Panel D) cells were determined. The effects of either no NAX038 or 100 nM NAX038 on the colony formation in the presence of different doses of metformin (MET) in AsPC-1 + pLXSN (Adh.) (Panel B) and BxPC-3 + pLXSN (Adh) (Panel E) were determined. The effects of either no NAX060 or 100 nM NAX060 on the colony formation in the presence of different doses of metformin (MET) in AsPC-1 + pLXSN (Adh.) (Panel C) and BxPC-3 + pLXSN (Adh) (Panel F) cells were determined. Different treated cell lines were normalized to untreated respective cell line. Statistical analyses were performed in comparison with BBR, NAX038 or NAX060 treated cells vs. metformin-alone treated cells where appropriate by the Student T test on the means and standard deviations of various treatment groups. *** = P < 0.0001, **P < 0.005, *P < 0.05, NS = not statistically significant. r> 2.2. Infection of cells with a retroviral vector encoding WT-TP53
    PDAC cell lines were infected with either a retroviral vector encoding WT-TP53 or the empty pLXSN vector as a control as described (Abrams et al., 2018, 2019). Stably infected cell lines were isolated in the presence of 2 mg/ml G418 (geneticin) Sigma-Aldrich. Pools were established after approximately four weeks in culture (Abrams et al., 2018, 2019).
    2.3. Methylthiazole tetrazolium assays
    Methylthiazole tetrazolium (MTT) assays were performed to determine the sensitivity of the pancreatic cancer cells to the modified NAX compounds and berberine as described (Abrams et al., 2018, 2019). To test the hypothesis that the various IC50s in the PDAC cells treated with the NAX compounds were statistically different than in the same cells treated the same day with the parental BBR, student's T tests were performed using Graph Pad Prism (QuickCals) statistical analysis.
    2.4. Colony formation assays
    Cells were seeded at 500 cells per well into three wells of six well Falcon plates (Falcon/Corning, Durham, NC, USA). Cells were treated with the indicated concentrations of chemotherapeutic drugs, metformin, BBR or NAX compounds. Colonies were propagated for approximately two weeks. When the colony size was approximately forty to 100 cells, the number of colonies were determined after giemsa staining of the colonies in the wells. The number of colonies were determined in three wells per condition and the average and standard deviation determined. The number of colonies in each condition were normalized to the untreated samples.