Molecular Mimics of Classic P-Glycoprotein Inhibitors as Multidrug Resistance Suppressors and Their Synergistic Effect on Paclitaxel

<div><p>P-glycoprotein (Pgp) is a membrane bound efflux pump spread in a variety of tumor cells and considered as a main component of multidrug resistance (MDR) to chemotherapies. In this work, three groups of compounds (imidazolone, oxazolone and vinyl dipeptide derivatives) were synthesized aiming to develop a molecular framework that effectively suppresses MDR. When tested for their influence on Pgp activity, four compounds coded Cur1-01, Cur1-12V, Curox-1 and Curox-3 significantly decreased remaining ATP concentration indicating Pgp substrate site blocking. On the other hand, Cur-3 and Cur-10 significantly increased remaining ATP concentration, which is indicative of Pgp ATPase inhibition. The cytotoxicity of synthesized compounds was examined against Pgp expressing/highly resistant colorectal cancer cell lines (LS-174T). Compounds Cur-1 and Cur-3 showed considerable cytotoxicity with IC₅₀ values of 7.6 and 8.9 μM, respectively. Equitoxic combination (at IC₅₀ concentrations) of PTX and Cur-3 greatly diminished resistant cell clone from 45.7% to 2.5%, albeit with some drop in potency from IC₅₀ of 7.9 nM to IC₅₀ of 23.8 nM. On the other hand, combination of PTX and the non-cytotoxic Cur1-12V (10 μM) significantly decreased the IC₅₀ of PTX to 3.8 nM as well as the resistant fraction to 16.2%. The combination test was confirmed using the same protocol but on another resistant CRC cell line (HCT-116) as we obtained similar results. Both Cur-3 and Cur1-12V (10 μM) significantly increased the cellular entrapment of Pgp probe (doxorubicin) elevating its intracellular concentration from 1.9 pmole/cell to 3.0 and 2.9 pmole/cell, respectively.</p></div

Isolation of Antiosteoporotic Compounds from Seeds of <i>Sophora japonica</i>

<div><p>Chemical investigation of <i>Sophora japonica</i> seeds resulted in the isolation of seven metabolites identified as: genistin (<b>1</b>), sophoricoside (<b>2</b>), sophorabioside (<b>3</b>), sophoraflavonoloside (<b>4</b>), genistein 7,4’-di-<i>O</i>-<i>β</i>-D-glucopyransoide (<b>5</b>), kaempferol 3-<i>O</i>-<i>α</i>–L-rhamnopyranosyl(1→6)<i>β</i>-D-glucopyranosyl(1→2)<i>β</i>-D-glucopyranoside (<b>6</b>) and rutin (<b>7</b>). Compounds <b>1</b>, <b>2</b> and <b>5</b> showed significant estrogenic proliferative effect in MCF-7 cell in sub-cytotoxic concentration range. Compounds <b>1</b> and <b>2</b> showed minimal cell membrane damaging effect using LDH leakage assay. Accordingly, compound <b>2</b> (sophoricoside, (SPH)) was selected for further <i>in-vivo</i> studies as a potential anti-osteoporosis agent. The anti-osteoporotic effect of SPH was assessed in ovarectomized (OVX) rats after oral administration (15 mg/kg and 30 mg/kg) for 45 days compared to estradiol (10 µg/kg) as a positive control. Only in a dose of 30 mg/kg, SPH regained the original mechanical bone hardness compared to normal non-osteoporotic group. However, SPH (15 mg/kg) significantly increased the level of alkaline phosphatase (ALP) to normal level. Treatment with SPH (30 mg/kg) increased the level of ALP to be higher than normal group. SPH (15 mg/kg) did not significantly increase the serum level of osteocalcin (OC) compared to OVX group. On the other hand, treatment with SPH (30 mg/kg) significantly increased the level of OC to 78% higher than normal non-ovarectomized animals group. In addition, SPH (15 mg/kg) decreased the bone resorption marker, acid phosphatase (ACP) to normal level and SPH (30 mg/kg) further diminished the level of serum ACP. Histopathologically, sophoricoside ameliorated the ovarectomy induced osteoporosis in a dose dependent manner. The drug showed thicker bony trabeculae, more osteoid, and more osteoblastic rimming compared to OVX group.</p></div

Cytotoxic Activity of synthesized compounds against LS174T CRC cell lines.

<p>Cytotoxic Activity of synthesized compounds against LS174T CRC cell lines.</p

Cytotoxicity assessment of compounds isolated from <i>S. Japonica</i> using trypan blue exclusion assay.

<p>Cytotoxicity assessment of compounds isolated from <i>S. Japonica</i> using trypan blue exclusion assay.</p

(A) The effect of test compounds on the activity of P-glycoprotein efflux pump cell free isolated recombinant P-gp protein and (B) within LS-174T cells.

<p>The influence of test compounds on the cellular pharmacokinetics via inhibiting the activity of Pgp pump. Verapamil (VRP) is a standard direct Pgp blocker. Sod Vanadate is a standard ATPase inhibitor. Curcumin is the prototype compound in the design. The asterisk (*) denotes significant difference from control.</p

Chemomedulatory effects of test compounds on the cytotoxicity parameters of PTX against LS-174T cell line.

<p>Chemomedulatory effects of test compounds on the cytotoxicity parameters of PTX against LS-174T cell line.</p

The effect of serial equitoxic concentrations (ratio 1:100) of Cur3 (A), and 10 μM (B), and serial concentration of Cur1-12V (ratio 1:100) (C) on the cytotoxicity of PTX in HCT-116 cells.

<p>Cells were exposed to serial dilution of PTX (●), Chemosensetizer (▼) or their combination (○) for 72 h. Cell viability was determined using SRB assay.</p

Examples of Pgp inhibitors.

<p>Examples of Pgp inhibitors.</p

Structure and codes of the synthesized oxazolones and imidazolones.

<p>Structure and codes of the synthesized oxazolones and imidazolones.</p

Chemical structure for compounds isolated from <i>Sophora japonica</i> seeds.

<p>Chemical structure for compounds isolated from <i>Sophora japonica</i> seeds.</p