Identification and Analysis of the Active Phytochemicals from the Anti-Cancer Botanical Extract Bezielle

Bezielle is a botanical extract that has selective anti-tumor activity, and has shown a promising efficacy in the early phases of clinical testing. Bezielle inhibits mitochondrial respiration and induces reactive oxygen species (ROS) in mitochondria of tumor cells but not in non-transformed cells. The generation of high ROS in tumor cells leads to heavy DNA damage and hyper-activation of PARP, followed by the inhibition of glycolysis. Bezielle therefore belongs to a group of drugs that target tumor cell mitochondria, but its cytotoxicity involves inhibition of both cellular energy producing pathways. We found that the cytotoxic activity of the Bezielle extract in vitro co-purified with a defined fraction containing multiple flavonoids. We have isolated several of these Bezielle flavonoids, and examined their possible roles in the selective anti-tumor cytotoxicity of Bezielle. Our results support the hypothesis that a major Scutellaria flavonoid, scutellarein, possesses many if not all of the biologically relevant properties of the total extract. Like Bezielle, scutellarein induced increasing levels of ROS of mitochondrial origin, progressive DNA damage, protein oxidation, depletion of reduced glutathione and ATP, and suppression of both OXPHOS and glycolysis. Like Bezielle, scutellarein was selectively cytotoxic towards cancer cells. Carthamidin, a flavonone found in Bezielle, also induced DNA damage and oxidative cell death. Two well known plant flavonoids, apigenin and luteolin, had limited and not selective cytotoxicity that did not depend on their pro-oxidant activities. We also provide evidence that the cytotoxicity of scutellarein was increased when other Bezielle flavonoids, not necessarily highly cytotoxic or selective on their own, were present. This indicates that the activity of total Bezielle extract might depend on a combination of several different compounds present within it

A convenient, rapid and eco-friendly synthesis of bis-indolylmethanes under microwave irradiation

327-330Silica chloride has been applied as a catalyst for the first time for rapid synthesis of bis-indolylmethanes in excellent yields by electrophilic substitution reaction of indoles with carbonyl compounds under microwave irradiation. The process is environmentally benign

Differential induction of ROS by Bezielle flavonoids.

<p><b>A.</b> Induction of peroxide type ROS by the flavonoids in MDAMB231 and MCF10A cells. Cells were treated for 30 minutes or 6 hours with the indicated flavonoids at 10 µg/ml and analyzed for green fluorescence (FL1) on a FACScan after loading with H₂DCF-DA. Results are shown as fold increase of the FL1 observed in untreated cells analyzed in same experiments (FL1 levels in untreated cells are marked by dotted line). <b>B.</b> Induction of mitochondrial superoxide by Bezielle flavonoids. After treatment as in A, cells were loaded with MitoSox Red and analyzed for levels of red fluorescence (FL2). Results are presented in the same way as in A, and are mean ± S.E. of three to five experiments <b>C.</b> Survival of MDAMB231 cells treated with flavonoids at 10 µg/ml for 24 hours in the absence or presence of 10 mM pyruvate or NAC. Results are mean ± S.E. (n = 3). Significant differences (** P<0.01, *P<0.05) between cells treated with flavonoids or Bezielle alone or in presence of antioxidants are e are shown. D. Same as in C, with MCF10A cells.</p

Selective cytotoxicity of scutellarein and its metabolic effects are increased when it is combined with other flavonoids.

<p><b>A.</b> Survival of cells treated with the scutellarein alone and combinations of flavonoids at the indicated ratios, all at a final concentration of 10 µg/ml. Mean ± S.E. (n = 3). <b>B.</b> Inhibition of OCR and ECAR by scutellarein and mixtures of flavonoids selectively in tumor cells. Cells were treated for 4 hours with scutellarein alone, or with the mixtures of scutellarein with apigenin and luteolin. Mean ± S.E. (n = 4). <b>C.</b> ATP levels in cells treated as in B. Results are mean ± S.E. (n = 3). Significant differences (** P<0.01, *P<0.05) between groups treated with mixtures of flavonoids versus scutellarein alone are shown.</p

Inhibition of mitochondrial respiration inhibits induction of ROS, DNA damage and cell death by flavonoids.

<p><b>A.</b> MDAMB231 and MDAMB231Rho-0 cells were treated with flavonoids for 6 hours, and levels of ROS and mitochondrial superoxide were quantified as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030107#pone-0030107-g002" target="_blank">Figure 2</a>. Results are mean ± S.E. (n = 3). <b>B.</b> MDAMB231 Rho-0 cells and MDAMB231 cells were treated with Bezielle, scutellarein or apigenin for 24 hours, and percentages of surviving cell quantified. Data are average of three experiments. <b>C.</b> Dissipation of mitochondrial transmembrane potential by flavonoids. Cells were treated for 2 hours, loaded with potential-sensitive dye JC-1 and analyzed by flow cytometry. The FL2 fluorescence is reduced in cells with lowered ΔΨM; FL2 of untreated cells was assigned a level of 100%. All treatments resulted in statistically significant dissipation of ΔΨM compared to control cells. Significant differences were also observed between cells treated with scutellarein and other groups, and are indicated (** P<0.01,*P<0.05). <b>D.</b> Detection of carbonylated proteins in cells treated with the indicated flavonoids or Bezielle for 4 hours. After treatment cells were fixed, treated with DNP, washed and incubated with a FITC-conjugated antibody to DNP, and analyzed by flow cytometry. All results are mean ± S.E. (n = 3). Significant differences (** P<0.01, *P<0.05) compared to untreated are shown.</p

Summary of the effects that individual flavonoids produce in tumor cells.

<p>Summary of the effects that individual flavonoids produce in tumor cells.</p

Effect of flavonoids on energy producing metabolic pathways.

<p><b>A.</b> Results of metabolic flux analysis of glycolysis (ECAR) and mitochondrial respiration (OCR) in MDAMB231 treated with flavonoids at 10 µg/ml or Bezielle at 300 µg/ml for 4 hours. Results are mean ± S.E. (n = 4). <b>B.</b> ECAR and OCR in MDAMB231 and MCF10A cells treated with 20 µg/ml of flavonoids for 4 hours. Results are mean ± S.E. (n = 4). <b>C.</b> ATP levels in cells treated as in B. Results are mean ± S.E. (n = 3). Significant differences (** P<0.01, *P<0.05) between treated and untreated groups are shown.</p

Bezielle flavonoids exhibit differential cytotoxicities towards cancer versus non-transformed cells.

<p><b>A.</b> Cytotoxic activity of apigenin (A), luteolin (L) and Bezielle in breast cancer cells MDAMB231 and non-transformed breast epithelial cell line MCF10A. Cells were treated for 24 hours prior to analysis with the concentrations of flavonoids indicated to the right of the charts (5, 10 and 20 µg/ml), or with Bezielle (BZL) at 250 µg/ml. <b>B.</b> Cytotoxic activities of scutellarein (S), isoscutellarein (IS) and carthamidin (C), as in <b>A.</b> All results are mean ± S.E. (n = 4).</p