Panel A: Representative histograms of MDA-MB-435 cells treated with 3mM CJ or MJ for 24 hr. Panel B: Quantitative graph of MDA-MB-435 cells treated with 0.5mM and 3mM CJ or MJ for 24 hr. Both CJ and MJ induced a significant G0/G1 block in MDA-MB-435 cells and 3mM MJ also induces S-block with increasing sub-diploid population. Panel C: Representative histograms of MCF-7 cells treated with 3mM CJ or MJ. Panel D: CJ and MJ induced G0/G1 block in MCF-7 cells and 3mM CJ also induced S-block while 3mM MJ increased sub-diploid population. (Two-tailed t-test, p<0.05). Results are representative of two different experiments.

Panel A: Western blot of caspase-8 in MDA-MB-435 cells treated with 3mM MJ for 2 hr, 4 hr and 8 hr. Procaspase-8 was processed and cleaved to active caspase-8 at all the time points studied. Caspase-8 activity was increased with 3mM MJ treatment in MDA-MB-435 cells compared to the control. β-actin was a loading control. Panel B: Fluoremetric assay showed increased caspase-8 activity with MJ treatment at 2 and 4 hr time points (p<0.01).

MAPK activity was measured as explained in methods. Panel A: In MDA-MB-435 cells increase in p38 phosphorylation was significant at 2hr (p<0.04) and starts decreasing from 4 hr till 8 hr. Panel B: In MDA-MB-435 cells significant increase in ERK1/2 phosphorylation was observed at 1hr (p<0.04). Panel C: In MCF-7 cells p38 phosphorylation was not observed at all time points studied. Panel D: In MCF-7 cells ERK1/2 phosphorylation was not observed at all time points studied. Results are representative of two independent experiments.

MJ activated caspase-3 in MDA-MB-435 cells. Panel A: Western blot of procaspase-3. Decreased levels of procaspase-3 were observed in MDA-MB-435 cells treated with 3mM MJ. Panel B: Caspase-3 activity measurement by flow cytometry showed activation of caspase-3 in MDA-MB-435 cells but not in MCF-7 cells. Fold increase was calculated with respect to the control (1). Results are representative of two different experiments. Panel C: Mitochondrial membrane potential was determined in breast cancer cells as explained in methods. In both MDA-MB-435 and MCF-7 cells IC50 MJ and 3mM MJ decreased the membrane potential as observed by an increase in green fluorescence compared to the control (p<0.001).

Increase in TNFR1 expression and MAPK activity was measured as explained in the methods section. Panel A: From left, first histogram represents TNFR1 expression in MDA-MB-435 cells treated with DMSO (0.3%). From left, second histogram represents TNFR1 expression in MDA-MB-435 cells treated with 1mM MJ for 24 hr. From left, third histogram represents TNFR1 expression in MDA-MB-435 cells treated with 2mM MJ. From left, fourth histogram represents TNFR1 expression in MDA-MB-435 cells treated with 3mM MJ. Fifth histogram represents TNFR1 expression in MDA-MB-435 cells treated with 500 μM DCA, which showed increase in TNFR1 expression. Increase in TNFR1 expression was dose dependent and at 3mM MJ a complete shift in fluorescence intensity was observed. Panel B: From left, first histogram represents TNFR1 expression in MCF-7 cells treated with DMSO (0.3%). From left, second histogram represents TNFR1 expression in MCF-7 cells treated with 1mM MJ for 24 hr. From left, third histogram represents TNFR1 expression in MCF-7 cells treated with 2mM MJ. From left, fourth histogram represents TNFR1 expression in MCF-7 cells treated with 3mM MJ. Fifth histogram represents TNFR1 expression in MCF-7 cells treated with 500 μM DCA, which showed increase in TNFR1 expression. Increase in TNFR1 expression was dose dependent and at 3mM MJ a complete shift in fluorescence intensity was observed. Panel C: MDA-MB-435 cells were exposed to 3 mM MJ in the presence of TNFR1 antibody. Percent viability was increased which indicated TNFR1 expression in breast cancer cells. Cells treated with TNFR1 antibody and control antibody served as negative controls.

Panel A-D: CJ and MJ inhibited the long-term proliferation of breast cancer cells. Panel A: MDA-MB-435 cells were exposed to varying concentrations of CJ or MJ for 24hr. Panel B: MCF-7 cells were exposed to varying concentrations CJ or MJ for 24 hr. Panel C: MDA-MB-435 and MCF-7 cells were exposed to 3 mM CJ for 0, 2, 4 and 8 hr. Panel D: MDA-MB-435 and MCF-7 cells were exposed to 3 mM MJ for 0, 2, 4 and 8 hr. At termination the drugs were washed off and the effects on long term cell proliferation were determined. Colonies (≥50 cells/colony) were counted and survival was calculated as percentage of control cells (100%). Values are Mean±SEM; results are representative of duplicate analyses of two separate experiments. CJ and MJ significantly inhibited the long-term proliferation of all two cell lines in dose and time dependent manner (p<0.001; one tailed t-test).

Cytotoxicity of CJ or MJ on human breast cancer cells (MDA-MB-435 and MCF-7) as well as on normal human cells (CDC-1070-SK and MCF-10A) in culture. Panel A: MDA-MB-435 and MCF-7 cells were exposed to varying concentrations of CJ for 24 hr. Panel B: MDA-MB-435 and MCF-7 cells were exposed to MJ for 24 hr. Panel C: CDC-1070-SK and MCF-10A were exposed to different concentrations of MJ for 24 hr. The effects on cell viability were determined by mitochondrial dehydrogenase assay using alamarBlue dye as described in the methods section. Cell viability was calculated as a percentage of untreated cells (100%). Values were Mean±SD (n=6); results are representative of three independent experiments. At concentrations 1.5mM and higher there was a significant decrease in the viability of breast cancer cells compared to the control. One tailed t-test was carried out and at concentrations of 1 mM and above, a statistically significant (p < 0.0001) decrease in viability was observed. MJ was not cytotoxic to CDC-1070-SK and MCF-10A cells (Panel C). Results are representative of three different experiments.

Panel A: To confirm apoptosis induction breast cancer cells were stained cells with Hoechst and fluorescence microscopy images were acquired. Percent apoptosis was calculated as explained in the methods section. Both CJ and MJ induced apoptosis in breast cancer cells. Results are representative of two different experiments (p<0.01). Panel B: MDA-MB-435 cells were treated with DCA (500μM) for 4 hr, 8 hr and 24 hr. DCA induced G2/M arrest at all three time points measured and apoptosis at 24 hr. Panel C: DNA gel photograph of MDA-MB-435 and MCF-7 cells treated with MJ for 24 hr. Lane 1: High DNA Mass™ Ladder shows 1000, 2000, 3000, 4000, 6000, 10000 bp bands. Lane 2: MDA-MB-435 cells treated with 0.03% DMSO (Control). Lane 3: MDA-MB-435 cells treated with 1mM MJ. Lane 4: MDA-MB-435 cells treated with 2mM MJ. Lane 5: MDA-MB-435 cells treated with 3mM MJ. Lane 6: MDA-MB-435 cells were treated with DCA for 4hr. Lane 7: MDA-MB-435 cells were treated with DCA for 8 hr. Lane 8: MCF-7 cells treated with 0.03% DMSO (Control). Lane 9: MCF-7 cells treated with 1mM MJ. Lane 10: MCF-7 cells treated with 2mM MJ. Lane 11: MCF-7 cells treated with 3mM MJ. DNA smear was observed only in MDA-MB-435 cells treated with 3mM MJ and DCA treated cells were used as positive controls. Results are representative of three different experiments.