Primary antibodies: rabbit anti-human PARP-1 (polyclonal, 1:1,000) and rabbit anti-human caspase-3 (monoclonal, 1:1,000) were from Cell Signaling Technologies (Boston, MA, USA). Mouse anti-human α-tubulin (monoclonal, 1:1,000) was from Sigma-Aldrich (St. Louis, MO, USA). Goat anti-human actin (polyclonal, 1:1,000) and mouse anti-human GAPDH (monoclonal, 1:100,000) were from Santa Cruz Biotechnology (Dallas, TX, USA). Secondary antibodies: peroxidase-conjugated goat anti-rabbit, goat anti-mouse and rabbit anti-goat IgG (H+L) antibodies were from Jackson (Baltimore Pike West Grove, PA, USA). Propidium iodide, MG132 (Z-Leu-Leu-Leu-al), insulin, doxorubicin, 5-fluorouracil, cholera toxin and hydrocortisone were from Sigma-Aldrich. DMEM F/12, high glucose DMEM, L-glutamine, donor horse serum, fetal bovine serum, recombinant human EGF, trypsin and PBS were from Biological Industries (Beit-Ha-Emek, Israel). Prostate epithelial growth medium was from Lonza (Walkersville, MD, USA).

Human MCF10A non-tumorigenic breast epithelial cells were propagated in DMEM F/12 medium supplemented with 5% horse serum, 2 ng/ml epidermal growth factor, 100 ng/ml cholera toxin, 50 ng/ml hydrocortisone and 10 μg/ml insulin. Human HCT116 colorectal carcinoma, PC-3 prostate adenocarcinoma, DU-145 prostate carcinoma, MCF7 breast adenocarcinoma and MDA-MB-231 breast adenocarcinoma cells were propagated in high glucose DMEM supplemented with 10% fetal bovine serum and 2 mM L-glutamine. All cell lines were from American Type Tissue Collection (ATCC, USA) and were authenticated using STR analysis. Human EP#2 non-tumorigenic normal prostate epithelial cells were donated by Dr Orit Leshem (7) and propagated in prostate epithelial growth medium. All cells were propagated in a 37°C humidified incubator with 5% CO₂.

A dry extract powder of the formula (Zen Herbs Inc.) was dissolved in PBS at a concentration of 100 mg/ml. The solution was then centrifuged at 4300 g for 5 min, with the supernatant filtered through a 0.45-μM Millex PVDF filter (Merck Millipore, Tullagreen, Ireland). Solubility was estimated by cryophilization and weighting of the pellet, and was estimated to be ~50%. For convenience, the final stock concentration was designated at 100 mg/ml (w/v concentration of crude powder in PBS), enabling the comparison of the individual herbal components with their variable solubilities with the formula in its entirety.

Breast, prostate and colorectal cells were plated in triplicate into 96-well plates (MCF10A at 6×10³/w; MCF7, DU-145 and HCT116 at 3×10³/w; PC-3 and MDA-MB-231 at 4×10³/w; EP#2 - 8×10³/w), and allowed to attach and grow overnight. The medium was replaced with a fresh treatment-containing medium, and the cells were propagated for an additional 72 h. Cell viability was determined by XTT cell proliferation kit (Biological Industries) by replacing the medium with a fresh medium (in order to prevent interference of treatment color with XTT signal), and the addition of an XTT for 2–3 h. The resulting signal was measured by Power Wave × 340-I ELISA reader (Biotek Instruments, Winooski, VT, USA), with each cell line tested in at least three independent assays.

Both cancer and non-tumorigenic cells were plated at a density of 0.6-1×10⁶/10 cm plate, and then treated the following day. The cells were collected by trypsinization into their own medium to prevent loss of dead cells, with each sample divided into two aliquots. The first aliquot was analyzed for necrosis following exposure to a free propidium iodide (PI) influx for 15 min. The second aliquot was fixed with 70% ice-cold ethanol, stained with PI and used for cell cycle and apoptosis analysis. Cell sorting was performed on a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Cells were resolved on an FL-2 logarithmic scale for necrosis analysis and on an FL-2 linear scale for apoptosis (cell cycle) analysis, and later analyzed using a WinMDI 2.9 program (Purdue University Cytometry Laboratories, West Lafayette, IN, USA).

Cancer and non-tumorigenic cells were plated at a density of 6×10⁵/10 cm plate. On the following day cells were exposed to LCS101 treatments as indicated in the legends. After 24–72 h, the cells were collected by scraping, washed with cold PBS and lysed with RIPA (150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, 0.5 M Tris pH 8.0), supplemented with complete mini protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). Protein concentration was determined with Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). Samples (50 μg) were resolved by 8% SDS-PAGE, transferred to Protran BA-83 0.2 μm nitrocellulose membrane (Whatman, Piscataway, NJ, USA), blocked with 5% skimmed milk and immunoblotted with appropriate antibodies. The membrane was then washed thrice with TBST, incubated with corresponding HRP-conjugated secondary antibodies, probed with EZ-ECL enhanced chemiluminescence detection kit (Biological Industries) according to the manufacturer’s instructions and then exposed to Fuji Super RX film (Fujifilm, Tokyo, Japan).

Breast cancer cells were plated at a density of 6×10⁵/10-cm plates and treated the following day with 3 mg/ ml LCS101. After 24 h the cells were collected by scrapping and washed with cold PBS. Total RNA was isolated using an RNeasy Mini kit (Qiagen, GmbH, Hilden, Germany). RNA concentration and quality were determined by optic density measurement (260, and 280 nm). The quality of the samples was further verified by electrophoresis on 1% agarose gel, stained with ethidium bromide to visualize the 18S and 28S rRNA bands. Complementary DNA (cDNA) was prepared using random primers and a High Capacity cDNA Reverse Transcription kit (AB Applied Biosystems, Foster City, CA, USA). cDNA was subjected to RT-qPCR on a StepOnePlus Real-Time PCR System using a Power SYBR Green PCR Master Mix (AB Applied Biosystems). The RT-qPCR was performed according to the manufacturer’s instructions using the following primer sets: PARP-1 forward, 5′-AAGCTCT ATCGAGTCGAGTACG-3′; reverse, 5-GAAGCTCAGAGA ACCCATCC-3. GADPH forward, 5-TGGACCTCATGG CCCACA-3; reverse, 5-TCAAGGGGTCTACATGGCAA-3. The expression levels of PARP-1 from triplicate reactions was determined by normalization to GAPDH according to the manufacturer’s instructions.

The mean ± standard deviations were calculated in each experiment, which were performed in triplicate. The data were collated and analyzed in a Microsoft Excel 2007 program.

Initially we treated the different tumor cell lines and non-tumorigenic human cell lines with the LCS101 compound. Exposure of the cultured tumor cells to the compound led to a dose-dependent reduction in cell viability, with cell death observed in >90% of cells, as measured by XTT assay. This phenomenon was observed at concentrations of 1 mg/ml for breast and colon cancer cell lines, and at 3 mg/ml for prostate cancer cell lines. At the same time, the non-tumorigenic human epithelial cell lines MCF10A (breast) and EP#2 (prostate) demonstrated a reduction in viability of <30% following exposure to the botanical compound (Fig. 1A). The non-tumorigenic human luminal breast cell line HB-2 also displayed an attenuated response to LCS101 exposure (not shown). Light microscopy showed increased cell death of all cancer cells following exposure to LCS101 treatment, with some of the treated cancer cells demonstrating swelled morphology indicative of a necrotic process. In contrast, non-tumorigenic human epithelial cells exhibited normal density and morphology following exposure to the compound (Fig. 1B).

To address the mechanisms underlying the anticancer activity of LCS101, MDA-MB-231, MCF7 and MCF10A breast cells were treated with LCS101, and expression of apoptosis markers caspase-3 (cas-3) and poly(ADP-ribose) polymerase 1 (PARP-1) were examined. In classic apoptosis both of these proteins undergo cleavage, which is considered as hallmark of apoptosis. Surprisingly, no cleavage of caspase-3 and PARP-1 was detected, though a significant reduction in the level of PARP-1 protein was observed in both of these cancer cell lines. In contrast, the non-tumorigenic human epithelial breast MCF10A cells exposed to LCS101 showed no reduction in PARP-1 levels (Fig. 2A).

In order to better understand the cytotoxic effects of the LCS101 formulature, 6 of the 14 herbal components were isolated and selected: Ligustrum lucidum, Milletia reticulata, Paeonia lactiflora, Paeonia obovata, Prunella vulgaris and Scutellaria barbata. These herbs were found to display greater toxic effects towards MDA-MB-231 cancer cells, without any harmful effects on the non-tumorigenic human epithelial breast MCF10A cells (not shown). Following these findings, the LCS101 formula was divided into the ‘toxic formula’ (the above-mentioned 6 components) and the ‘non-toxic formula’ (the remaining 8 components). Exposure to the toxic formula resulted in a significant increase in cell death in MCF7 and MDA-MB-231 breast cancer lines, compared to no cytotoxic effect in the non-toxic formula (Fig. 2B and C). Following exposure to the toxic formula, cell swelling was observed in treated MDA-MB-231 cells (Fig. 2C, arrows). Both the toxic and non-toxic formulas had no effect on the non-tumorigenic human epithelial breast MCF10A cells (Fig. 2B and C). Finally, while the toxic formula reduced PARP-1 levels in MDA-MB-231 cells, no such effect was observed with the non-toxic components of the formula (Fig. 2D).

To further address the mechanism of PARP-1 protein reduction, we tested PARP-1 mRNA level in LCS101-treated cells using RT-PCR. PARP-1 mRNA levels in exposed MCF7 and MDA-MB-231 cells were similar to those found in controls (Fig. 3A). We then examined PARP-1 protein degradation using the proteasome inhibitor MG132 on MDA-MB-231 cells. After 24 h of treatment with MG132, which was necessary for LCS101-induced PARP-1 reduction, massive cell death and complete PARP-1 cleavage, characteristic of apoptosis, were observed. However, the examination of MCF7 cells, which apparently were more resistant to MG132-induced apoptosis, found that the MG132 failed to prevent PARP-1 elimination following exposure to the botanical formula (Fig. 3B).

LCS101-treated cells were analyzed using FACS in order to distinguish between apoptotic and necrotic features. For this purpose, MCF7, MDA-MB-231 and MCF10A cells were treated with LCS101 for 72 h, with each sample divided into two aliquots for FACS analysis. One aliquot was fixed and stained with PI to assess the sub-G1 population, which contained cells with degraded DNA, characteristic of apoptotic death. The second aliquot was used to evaluate the percentage of cells with ruptured membranes, typical of necrotic death, using free PI influx by live unfixed cells. Ruptured membrane of necrotic cells allows free PI uptake which causes the necrotic population to appear very bright on logarithmic PI scale. Following exposure to LCS101, the pattern of the vast majority of the cancer cell lines MCF7 and MDA-MB-231 moved far to the right upon free PI uptake, indicating that most of the cells possessed a necrosis-like ruptured membrane (Fig. 4). Morphologically, a number of the affected cells exhibited significant swelling, typical of a necrotic process as well (Fig. 1B). At the same time, only 20% of the cells exhibited sub-G1 DNA content, a typical indicator of apoptosis (Fig. 4). LCS101 did not induce cell death in the non-tumorigenic human epithelial breast MCF10A cells (Fig. 4).

Chemotherapy-induced cell death was observed in all three breast cell lines (MCF-10A, MCF-7 and MDA-MB-231) following exposure to the chemotherapy agent doxorubicin (Fig. 5A). When introduced at a concentration of 3 mg/ml, LCS101 augmented the tumoricidal effects of the chemotherapy in MCF7 and MDA-MB-231 cells. In contrast, the addition of the botanical formula to the non-tumorigenic human epithelial breast MCF10A cells greatly reduced cell death (Fig. 5A and C). A similar selective effect was seen in 5-FU-treated cell lines, with LCS101 increasing the tumoricidal effect of the chemotherapy agent on MCF7 and MDA-MB-231 cells, while reducing cell death in the non-tumorigenic human epithelial breast MCF10A cells (Fig. 5B). In addition to the above findings, MDA-MB-231 cells treated with doxorubicin demonstrated PARP-1 cleavage typical of apoptosis, at 48 and at 72 h (Fig. 6A). A less prominent but still clearly detectable PARP-1 cleavage was observed in MCF10A and MCF7 cells at 72-h treatment with doxorubicin (Fig. 6B). The addition of LCS101 to doxo-rubicin-treated MDA-MB-231 and MCF7 cells, however, led to the disappearance of PARP-1. In non-tumorigenic human epithelial breast MCF10A cells, LCS101 prevented doxorubicin-induced PARP-1 cleavage altogether, supporting our previous findings showing protective effect of LCS101 in normal cells.

In order to confirm the above findings, we performed FACS analysis of MDA-MB-231, MCF7 and MCF10A cells, which were treated with both doxorubicin and LCS101 (Fig. 4). Our findings were consistent with those above regarding PARP-1 cleavage, with typical apoptosis observed in all three cell lines treated with doxorubicin alone and a clearly demarked sub-G1 apoptotic population. At the same time, the addition of LCS101 reduced apoptosis in non-tumorigenic human epithelial breast MCF10A cells, while increasing cell death in the two cancer cell lines (Fig. 4). In cancer cells treated with both LCS101 and doxorubicin, cell death exhibited necrotic features, as described above. This indicates that the increase in doxorubicin-induced cell death in cancer cell lines treated with LCS101 results from a necrosis-like process. At the same time, LCS101 offers a protective effect on non-tumorigenic cells exposed to the chemotherapy agent.