Identification of isolated compounds

The aerial parts (bark, twigs, and leaves) of A. hydaspica were collected and subjected to extensive extraction and fractionation as described in the Materials and Methods section. Diagrammatic illustration of the process is shown in Supplementary Fig. 1. Structures of purified compounds were elucidated by 1D and 2D NMR and mass spectrometric analysis (ESI and APCI). The compounds isolated from A. hydaspica were identified by comparison of the physical data with those reported previously. The data indicated purification of the polyphenol methyl gallate (MG)20 and three flavan-3-ols (+) 7-O-galloyl catechin (GC)21, (+) catechin (C)22,23, and (+) catechin-3-O-gallate (CG)24; chemical structures of the isolated compounds are shown in Fig. 1.

Figure 1: Structures of isolated compounds from A. hydaspica R. Parker. Chemical structures of the various compounds purified from A. hydaspica are shown. Full size image

Effects of AHC compounds on cell viability

To investigate the effects of AHCs on cancer cell viability, PC-3 (an androgen-independent prostate cancer cell line) and MDA-MB-231 (a triple negative breast cancer cell line) were chosen as two representative cell lines. PC-3 and MDA-MB-231 cells were grown in 96-well plates and treated with AHCs at concentrations varying from 0 μM to 100 μM. To assess the effects of these compounds, we employed the Cell Titer 96® Aqueous One assay. The assay involves conversion of a MTS tetrazolium compound to a colored formazan product whose absorbance is directly proportional to the number of metabolically active cells and thus indirectly measures cell growth. As shown in Fig. 2, GC, C, CG and MG retarded PC-3 cell growth in a dose- and time-dependent manner as compared with untreated or DMSO treated control groups. The relative viability after 24 h of treatment with 100 μM doses of GC and C was 45.9 ± 3.7 and 40.5 ± 2.5%, respectively. The viability of cells decreased further after 48 h and 72 h of treatment. Similarly, the percentages of viable cells after 24 h treatment with 50 μM CG and MG were 45.5 ± 3.1% and 45.8 ± 2.3%, respectively, and viability continued to decrease through 72 h of treatment (Fig. 2). The well-known CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) was used as a positive control; however, it reduced viability in PC-3 cells to a lesser extent than the AHCs at similar concentrations.

Figure 2: Cell viability in cells treated with AHC compounds. (a–d) Prostate cancer PC-3 cells were treated with varying concentrations of (a) 7-O-galloyl catechin, (b) catechin, (c) catechin gallate, or (d) methyl gallate for 24, 48 and 72 h as indicated. TBB was included as a positive control and DMSO was included as a negative control at a concentration equivalent to 100 μM AHC compound. Cell viability was determined relative to the untreated (0 μM) cells. (e,f) Breast cancer MDA-MB-231 cells were treated with varying concentrations of (e) catechin gallate, or (f) methyl gallate for 24 or 48 h, as indicated. All other conditions are as describe for (a–d). Data are presented as mean ± SEM (n = 3). Data analyzed by two-way ANOVA with Bonferroni post-test. The asterisks *, **, ***indicate significant difference at p < 0.05, p < 0.001 and p < 0.0001, respectively, from the untreated control cells. Full size image

The cell viability studies demonstrated that CG and MG were the most potent anti-proliferative compounds in MDA-MB-231 cells, producing both dose- and time-dependent effects (Fig. 2). Cell viability with 50 μM CG was 56.0 ± 3.0% and 15.5 ± 1.2%, following 24 h and 48 h of treatment, respectively. The percent survival after 24 h and 48 h treatment of MDA-MB-231 cells with 50 μM MG was 54.6 ± 2.8% and 29.5 ± 4.0%, respectively. Lower doses (12.5 and 25 μM) also showed some cytotoxicity at 48 h of treatment. The effects of 100 μM TBB on cell viability were similar to those observed with 50 μM CG and MG. GC and C had relatively little effect on these cells (data not shown).

Detection of cell death by morphological analysis

In order to determine the role of apoptosis in cell growth inhibition by AHCs, morphological changes in PC-3 cells and MDA-MB-231 cells were examined by phase contrast microscopy (Supplementary Fig. 2). PC-3 cells were treated with 100 μM of GC and C, and 50 μM of CG and MG; MDA-MB-231 cells were treated with 50 μM of CG and MG. Phase contrast microscopy showed that treatment with AHCs for 24 and 48 h in both cell lines resulted in low cell confluence and membrane blebbing, indicative of apoptosis. Moreover, floating cells indicated that AHC treatment resulted in reduced adherence. Untreated or DMSO-treated (at volume equivalent to that for 100 μM AHC) cells were attached to the culture plates with greater than 90% confluence under the same conditions as those for AHC treated cells (Supplementary Fig. 2).

Fluorescence microscopy analysis of DAPI stained cells was undertaken to study nuclear alterations and apoptotic body formation, both of which are also features of apoptosis25. Cells treated with AHCs exhibited apoptotic morphology in both types of cancer cells under investigation. Morphological changes characterized by cytoplasmic and nuclear shrinkage, chromatin condensation and apoptotic body formation were observed in response to treatment of both PC-3 (Fig. 3a) and MDA-MB-231 (Fig. 3b) cells with AHCs. In the untreated or DMSO treated PC-3 and MDA-MB-231 cells, the stained nuclei were rounded and homogeneously stained with DAPI, whereas treated cancer cells from both cell lines showed an altered nuclear DNA staining pattern with condensed chromatin and apoptotic bodies that are hallmarks of early and late apoptosis.

Figure 3: Detection of apoptosis by fluoresence microscopy in cells after treatment with AHC compounds. (a) PC-3 cells were treated with GC, CG or MG for 48 h, as indicated below the panels. Cells were stained with DAPI, acridine orange and ethidium bromide (AO/EB), or propidium iodide (PI) as indicated on the right side of the panels. (b) MDA-MB-231 cells were treated with CG or MG for 48 h as indicated below the panels. Cells were stained with DAPI, acridine orange and ethidium bromide (AO/EB), or propidium iodide (PI) as indicated on the right of the panels. Images of cells were captured at 200-fold magnification. Scale bar is 100 μm. Full size image

The results of acridine orange (AO) and ethidium bromide (EB) staining of cells treated with AHCs are shown in Fig. 3. AO is a cell permeable fluorescent dye and stains nuclear DNA in both live and dead cells, whereas EB is a fluorescent dye that only stains nuclear DNA in cells that have lost their membrane integrity26. We observed that after AO/EB staining viable cells were uniformly stained green, early apoptotic cells were stained greenish yellow or displayed green yellow fragments, late apoptotic cells were stained orange or displayed orange fragments, and necrotic cells showed orange to red fluorescing nuclei with no indication of chromatin fragmentation. In PC-3 cells, treatment with GC (100 μM) showed late apoptotic cells that were densely stained as orange. Treatment with C (100 μM) showed live cells with initial phases of apoptotic nuclei appearing as “viable apoptotic” (VA) as indicated by green chromatin which was highly condensed or fragmented with some cells undergoing cell death. Dead cells also showed apoptotic nuclei as chromatin appeared to be fragmented indicating cells to be “nonviable apoptotic” (NVA). CG and MG were more effective in inducing cell death as compared with GC and C in PC 3 cells, and the mode of cell death was apoptosis indicated by highly fragmented and condensed chromatin which appeared bright orange (Fig. 3a). Similar results were obtained for MDA-MB-231 cells treated with various compounds (Fig. 3b).

Propidium iodide (PI) is a red fluorescent dye that is impermeable to the cell membrane of viable cells, and is used to stain the DNA of both necrotic and apoptotic cells. After 48 h of AHC treatment, untreated or DMSO treated cells showed no significant change in their viability as indicated by the very low number of cells staining with PI. In contrast, all tested compounds induced significant cell death as indicated by PI staining in PC-3 and MDA-MB-231 cells (Fig. 3).

AHCs induce loss of clonal survival

In order to assess the survival and proliferative capacity of the PC-3 and MDA-MB-231 cells following treatment with AHCs, clonal survival assays were carried out. In these assays, the AHCs were added to the cells and then removed after a 48 h period of treatment. The cells were counted and re-plated in complete media as described under Materials and Methods. After 7 days of growth, the cells were stained with crystal violet and colonies of 50 or more cells were counted. Representative stained colony plates are shown in Fig. 4; also depicted in Fig. 4 is the plot showing quantitation of the survival data for PC-3 and MDA-MB-231 cells. As shown, MDA-MB-231 and PC-3 cell colony numbers were significantly reduced (p < 0.001) compared to untreated or DMSO treated control groups. In PC-3 cells, cell shrinkage and a shift from stellate to a rounded appearance was more obvious as compared to MDA-MB-231 cells. We also observed small scattered cells unable to colonize and reduced colony size in both cancer cell types.

Figure 4: Clonogenic survival assays following treatment with AHC compounds. (a,b) PC-3 (a) or MDA-MB-231 (b) cells were treated with AHC compounds or equal dilution DMSO (as indicated below the plate images). After 48 h, the compounds were removed and cells seeded at a density of 1000 cells for PC-3 and 500 cells for MDA-MB-231 on 35 mm plates. After 7 days of growth, the cells were stained with crystal violet and the stained plates scanned. Representative plates are shown. (c) Crystal violet stained colonies were quantified as described in the “Materials and methods” section. The percent inhibition of PC-3 and MDA-MB-231 colony formation relative to untreated control is graphed. Data are presented as mean ± SEM of at least 3 independent experiments. Data analyzed by one-way ANOVA with Bonferroni post-test. Asterisks ***indicate significant difference at p < 0.0001 relative to untreated control. Full size image

Signaling pathways and survival proteins modulated by AHC treatment

Many of the chemopreventive agents are known to exert moderate inhibitory effects on diverse survival signaling pathways (e.g.,9). Thus, we decided to examine the effects of AHCs on certain survival signaling pathways in prostate and breast cancer cells. For these studies, we performed western blot analyses of lysates from PC-3 cells treated with 100 μM GC and C, and 50 μM CG and MG, and from MDA-MB-231 cells treated with two doses of compounds CG and MG (12.5 μM and 50 μM) for 24 or 48 h. For both cell lines, the signals analyzed were CK2α, CK2α′, PI3K, JAK2, Akt, Akt P-Ser473, STAT3, STAT3 P-Tyr705, ERK 1/2, ERK 1/2 P-Thr202/P-Tyr204, NFκB p65, NFκB p65-P-Ser529, IκBα P-Ser32/36, Bcl-2, Bcl-xL, xIAP, and survivin. The data shown in Fig. 5 (for PC-3 cells) and Fig. 6 (for MDA-MB-231 cells) are only for the signals that demonstrated changes in response to treatment with various agents (signals that were unchanged are not included); however, the quantitation of all signals (responsive as well as unresponsive) at 24 h and 48 h of treatment is shown in Supplementary Tables 2 and 3.

Figure 5: Western blot analyses following treatment of PC-3 cells with AHC compounds. Western blot analysis of cellular lysates prepared from PC-3 cells treated with 7-O-galloyl catechin (GC), catechin (C), catechin gallate (CG), methyl gallate (MG), or equal dilution of DMSO was performed. Treatments are indicated above the blots. The protein detected is indicated to the left of each blot, and the size of protein detected is indicated to the right of each blot. The quantitation of the data is shown in Supplementary Table 2. Cropped blots are shown. Full sized blots are included in Supplementary Fig. 3. All gels and blots were run under the same experimental conditions as described in Methods. Full size image

Figure 6: Western blot analyses following treatment of MDA-MB-231 cells with AHC compounds. Western blot analysis of cellular lysates prepared from MDA-MB-231 cells treated with 2 concentrations each of catechin gallate (CG) and methyl gallate (MG), or dilution of DMSO representing the highest concentration was performed. Treatments are indicated above the blots. The protein detected is indicated to the left of each blot, and the size of protein detected is indicated to the right of each blot. The quantitation of the data is shown in Supplementary Table 3. Cropped blots are shown. Full sized blots are included in Supplementary Fig. 4. All gels and blots were run under the same experimental conditions as described in Methods. Full size image

Polyphenolic compounds such as EGCG and resveratrol are known to exert an inhibitory effect on protein kinase CK2 in prostate cancer cells27. Thus, we examined the effects of these AHCs on CK2 and certain other signals in both prostate and breast cancer cells. Treatment of PC-3 cells with AHCs did not alter the expression levels of CK2α or α′ (Supplementary Table 2). Further, addition of AHCs to kinase activity assays did not lead to a dose dependent effect using purified CK2 (data not shown). However, in breast cancer cells, there was a dose-dependent effect of CG and MG on CK2α level but not of CK2α′ (Supplementary Table 3 and Fig. 6). PI3K signals in PC-3 cells were also not affected by various AHCs, but CG and MG exerted a dose-dependent effect in breast cancer cells (Supplementary Table 3). JAK2 was significantly inhibited by the AHCs tested in PC-3 cells and both CG and MG were potent inhibitors at 50 μM concentration in MDA-MB-231 cells. Akt was not inhibited by GC and C but showed significant reduction in the presence of CG and MG in prostate cancer cells, and significant reductions were also noted in the Akt-P-Ser473 and P-Thr308 signals (Supplementary Table 2). However, these signals did not show any change in response to CG and MG in breast cancer cells (Supplementary Table 3). STAT3 and STAT3-P-Tyr705 were unresponsive to the AHCs tested in PC-3 cells (Supplementary Table 2), whereas these signals showed significant reductions in MDA-MB-231 cells in response to the AHCs tested (Supplementary Table 3). CG at 50 μM had a moderate effect on ERK 1/2 but the corresponding ERK 1/2-P-Thr202/P-Tyr204 demonstrated a dramatic decrease in response to all the AHCs tested in PC-3 cells (Supplementary Table 2). On the other hand, the breast cancer cells did not demonstrate any significant effect on these two signals in response to CG and MG (Supplementary Table 3). NFκB p65 was unaffected by the various AHCs in PC-3 but there was a significant reduction in the p65-P-Ser529 signal; the IκBα P-Ser32/36 signal was markedly reduced in the PC-3 cells in response to the AHCs and was analogous to the reduction of p65 phosphorylation-specific signal (Supplementary Table 2). In the MDA-MB-231 cells, reduction of p65-P-Ser529 was concordant with that of the p65 in the presence of 50 μM CG or 50 μM MG (Supplementary Table 3). The effect on Bcl-2 in PC-3 cells was apparent in the presence of CG and MG (both at 50 μM concentrations) but not GC and C (at 100 μM) (Supplementary Table 2). Bcl-xL signal was reduced and demonstrated varying levels of response to different AHCs in both the prostate and breast cancer cells. The AHCs did not produce a significant change in level of xIAP in PC-3 or MDA-MB-231 cells, as compared to the change induced by TBB treatment. Finally, all the AHCs tested produced a significant loss of survivin signal in both the PC-3 and MDA-MB-231 cells (Supplementary Tables 2 and 3). These results suggested that AHCs result in varied effects on diverse survival signals in the prostate and breast cancer cells dependent on the type and dose of AHC.