We have determined that BZL101, an herbal mixture extracted from the Scutellaria barbata plant, is able to arrest the proliferation of hormone sensitive and insensitive human breast and prostate cancer cells that are considered representative models for early and late stage cancers. Our results demonstrate that BZL101 exerts phenotype specific molecular changes of cell cycle and steroid receptor gene expression in these human reproductive cancer cells. As summarized in , the specific phase of cell cycle arrest varied based on the individual characteristics of each cell line. In the less metastatic early stage hormone sensitive cell lines tested, MCF7 breast cancer cells and LNCaP prostate cancer cells, BZL101 arrested cell growth in the regulatory phases of the cell cycle; MCF7 cells arresting in the G1 phase of the cell cycle and LNCaP cells arresting in the G2/M phase of the cell cycle. In contrast, in both of the hormone insensitive cell lines that represent late stage cancers, MDA-MB-231 breast cancer cells and PC3 prostate cancer cells, BZL101 induced an S-phase arrest of the cell cycle. Furthermore, BZL101 was effective at slightly lower concentrations in the cell lines representing early stage cancers, 2 mg/mL for MCF7 cells and 1.5 mg/mL for LNCaP cells, as compared to the slightly higher concentration of 3 mg/mL necessary for arrest in the highly metastatic cancer cells MDA-MB-231 and PC3. In all cell lines tested, BZL101 increased the sub-G1 DNA content, which is indicative of cell death.
Figure 8 Proposed Model for the BZL101 disruption of cell cycle progression in hormone sensitive and insensitive breast and prostate cancer cell lines. BZL101 blocks cell cycle progression at different phases of the cell cycle that differ depending on the reproductive (more ...)
Consistent with the nature of the observed cell cycle arrests, BZL101 regulated the expression of cell cycle regulatory components that function in the corresponding phases of the cell cycle in each of the tested reproductive cancer cell lines (illustrated in ). For example, BZL101 strongly downregulated the protein production, transcript expression and/or promoter activities of the G1
-acting CDK2, CDK4 and cyclin D1 in MCF7 cells, which underwent a G1
cell cycle arrest. Cyclin B1 initiates G2
/M progression by promoting spindle assembly in mitosis,28
and BZL101 treatment of LNCaP cells induces a G2
/M cell cycle arrest with a corresponding loss of cyclin B1 expression. Cyclin A and CDK1 protein levels and transcript expression were strongly downregulated by BZL101 in both of the late stage cancer cell lines that undergo an S-phase cell cycle arrest. In each of the cell lines, BZL101 also altered the expression of one or more cell cycle components not directly associated with the phase of the cell cycle arrest. For example, all four cell lines showed a loss of CDK1 transcripts, while MDA-MB-231 and PC3 that undergo a S phase arrest display a significant downregulated expression of the G1
-acting CDK2 and CDK4. These results suggest that BZL101 activates transcriptional pathways common to each of the reproductive cancer cell lines. One unusual feature of our observations is that BZL101 signaling selectively regulates the expression of cell cycle components, such the CDKs, which in most instances do not display significant changes in their expression in response to anticancer compounds. We proposed that individual molecules within BZL101 disrupt the function of transcription factors that ultimately target the gene expression of the regulated cell cycle components.
BZL101 ablated ERα and AR expression in estrogen sensitive MCF7 breast cancer cells, and androgen sensitive LNCaP prostate cancer cells, respectively. This disruption of steroid receptor expression implicates BZL101 as a potential therapeutic in the treatment of relatively early stage hormone sensitive breast and prostate cancers. For ERα, deletion analysis of the promoter in MCF7 cells narrowed the functional BZL101 regulated region to a relatively small segment of the promoter that contains consensus DNA elements for the Prx2 transcription factor homolog S8 as well as for HNF3b (FoxA2), GATA and NKX-2 transcription factors. These transcription factors are through to have a roles in breast cancers cell proliferation and/or developmental processes.21–27
Regardless of the nature of the precise BZL101-regulated transcription factor(s), our results suggest that within the BZL101 mixture, specific compounds act as selective estrogen receptor disruptors. We further observed that treatment of MCF7 cells strongly downregulated expression of both EGFR and the ErbB2/HER2 member of the EGF receptor gene family. BZL101 was previously demonstrated to be effective in inhibiting proliferation of the HER2+
hormone insensitive SKBR3 breast cancer cell line,17
and is well tolerated in metastatic breast cancer patients with prior chemotherapeutic exposure.18
We now demonstrate that BZL101 can affect expression levels of HER2 in human breast cancer cells in an early stage cancer cell phenotype.
We have established that BZL101, an aqueous extract from the Scutellaria barbata plant, is an effective anticancer agent capable of inducing cell cycle arrest and increasing cell death of hormone sensitive and insensitive, early and late stage, breast and prostate cancer cell lines.
The chemical composition of BZL101 is complex29–31
and has been shown to contain several anticancer phytochemicals such as apigenin, luteolin and resveratrol,29,32
that can disrupt multiple proliferative pathways. In addition, this herbal mixture was recently shown to contain several anticancer flavones, such as Wogonin, Baicalein and Baicalin, which have both cytostatic and cytotoxic activities in certain cultured cells and can inhibit tumor growth in vivo.33
Intriguingly these flavones have no significant toxic effects on non-cancerous cells. It is tempting to consider that one more of these compounds mediate the ability of BZL101 to inhibit proliferation of a wide range of human reproductive cancer cells representing both early and late stage cancers.
Our studies suggest that individually, exposure of the early and late stage reproductive cancer cell line models used in our study to apigenin and luteolin, which are both found in BZL101, have only very modest to no effects on cell cycle control (data not shown). Conceivably, individual cell lines react with differing sensitivity to combinations of components of BZL101, leading to an overall decrease in proliferation and increased cellular death. We are in the process of determining which combinations of BZL101 containing molecules mediate the selective effects on cell cycle control that are phenotype dependent. The cell cycle arrest and steroid receptor disrupting properties of BZL101 shows the value of this plant extract as a promising therapeutic strategy, which, when combined with conventional treatments for reproductive cancer, could increase efficacy of treatment with reduced toxicity to patients.