Protein kinase C inhibitor chelerythrine selectively inhibits proliferation of triple-negative breast cancer cells

Triple-negative breast cancer (TNBC) is a subtype of breast cancer lacking targeted therapy currently. Recent studies imply that protein kinase C may play important roles in TNBC development and could be a specific target. In this study, we evaluated the anti-proliferative activity of PKC inhibitor chelerythrine on a panel of breast cancer cell lines. Chelerythrine selectively inhibited the growth of TNBC cell lines compared to non-TNBC cell lines as demonstrated by in vitro cell proliferation assay and colony formation assay, as well as evidenced by in vivo xenograft assay. The selective anti-proliferative effect of chelerythrine was associated with induction of apoptosis in TNBC cell lines. We further demonstrated that PKN2, one of the PKC subtypes, was highly expressed in TNBC cell lines, and knocking down PKN2 in TNBC cells inhibited colony formation and xenograft growth. This indicates that PKN2 is required for the survival of TNBC cells, and could be the target mediates the selective activity of chelerythrine. Finally, combination of chelerythrine and chemotherapy reagent taxol showed synergistic/additive effect on TNBC cell lines. Our results suggest chelerythrine or other PKC inhibitors may be promising regimens for TNBC tumors.

In vitro cell proliferation assay (SRB assay). The anti-proliferative effects of tested chemicals on breast cancer cell lines were assessed by sulforhodamine B (SRB) colorimetric assay as previously described 27 . Briefly, cells were seeded in 96-well plates in a volume of 100 μl/well at densities of 5,000~40,000 cells per well. After overnight incubation at 37 °C in a humidified incubator with 5% CO 2 , 100 μl medium containing chemicals (2 X indicated concentrations) were added. After treatment for 72 hours, attached cells were fixed with 50 μl cold 50% (w/v) trichloroacetic acid (TCA) for 1 hour at 4 °C and then stained with 100 μl 0.4% (w/v) SRB. The absorbency at 515 nm was measured using SpectraMax 190 microplate reader (Molecular Devices) after solubilizing the protein-bound dye with 200 μl 10 mM Tris base solution (pH 10.5). The IC50 value was defined as the concentration required for a 50% reduction in cell growth.
Colony formation assay. Cells were either treated with 5 μM chelerythrine for various periods of time or selected with puromycin for 3 days after lentivirus transduction. Cells were then washed with PBS, plated in drug-free medium in 6-well plates at densities of 1,000 cells/well and incubated for 7-10 days in the absence of drug. Colonies were stained with 0.2% (w/v) crystal violet in buffered formalin for 10 minutes. The number of colonies was counted.
Flow-cytometric analysis of cell cycle. Flow cytometric analyses were performed to define the cell cycle distribution after treatment with 5 μM chelerythrine. Cells were harvested, washed twice with PBS, resuspended in 0.5 ml PBS (1,000,000~2,000,000 cells/ml). Then 4 ml 70% ethanol was added and kept at −20 °C for 2 hours to fix the cells. Cells were stained for total DNA content with a solution containing 20 μg/ml propidium iodide, 200 μg/ml DNase-free RNase A, and 0.1% triton X-100 in PBS for 30 minutes at room temperature. Cell cycle distribution was analyzed by flow cytometry (BD Bioscience). The percentage of the total cell population in the four different phases (Sub-G0/G1, G0/G1, S, G2/M) of cell cycle was determined using FlowJo software.

Flow-cytometric analysis of apoptosis. Cellular apoptosis was analyzed with BD Annexin V:
Fitc Apoptosis Detection Kit I (BD Bioscience) by flow cytometry. Briefly cells were plated in 6-well plates (100,000~400,000 cells/well) and treated with chelerythrine. At the indicated time point, cells were harvested, washed twice with cold PBS, and resuspended in 1X Binding Buffer at a concentration of 1,000,000 cells/ml. 100 μl cells were transferred to 1.5 ml conical tube and 5 μl FITC Annexin V and 5 μl propidium iodide were added. The mixture was gently vortexed and incubated at room temperature for 15 minutes in the dark, followed by adding 400 μl 1x Binding Buffer to each tube. Cells were filtered and analyzed by flow cytometry (BD Bioscience) within 1 hour. Total apoptotic cells (FITC Annexin V positive) were counted. Assessment of cell morphological changes. Cells were plated in 12-well plates (80,000-200,000 cells/ well) then treated with 5 μM chelerythrine for 24 hours. After incubation, cells were collected, washed with PBS and stained with Hoechst 33258 (11.1 μg/ml) in buffered formalin solution containing 5.6% NP-40. Living and apoptotic cells were visualized through DAPI filter of fluorescence inverted microscope (Leica DM2500 Fluorescence Microscope) at ×400 magnification.
Lentivirus mediated gene knockdown. The following double strand oligos (only sense strands indicated) were cloned into the pLKO.1 plasmid at AgeI and EcoRI sites and sequence verified before use. shPRKCA: 5′-CCGGCGAGGTGAAGGACCACAAATTCTCGAGAATTTGTGGTCCTTCACCTCGTTTTTTG-3′, shPKN2: 5′-CCGGGTCCACGTCAAAGTATGATATCTCGAGATATCATACTTTGACGTGGACTTTTTG -3′. A MISSION non-target shRNA control vector served as the scrambled control (Sigma-Aldrich, SH002). Lentivirus were produced by cotransfection of 293T cells with above constructs and the MISSION packing mix (Sigma-Aldrich) using FuGENE HD transfection reagent (Promega). Cells were incubated with lentivirus for 24 hours before selection with puromycin (Gibco). Gene knockdown was confirmed by western blotting analysis.
Retrieval of gene expression data from CCLE database. Cancer Cell Line Encyclopedia (CCLE) data on breast cancer cell lines was used to compare mRNA expression of TNBC and non-TNBC cells 28 . The log 2 radio of TNBC cell lines (n = 26) versus non-TNBC cell lines (n = 30) was analyzed using GENE-E and Prism software. Quantitative real-time PCR. Cellular mRNA was purified by binding to poly(dT) magnetic beads (Dynal) and reverse transcribed using SuperScript III (Invitrogen) as described by the manufacturer. Quantitative real-time PCR was performed in duplicates three times by using SYBR Green (Molecular Probes) on the ViiA ™ 7 Real-Time PCR System (Applied Biosystems). Data were expressed as relative mRNA levels normalized to the eukaryotic translation initiation factor (EIF3S5 or TIF) expression level in each sample. The primer sequences can be obtained upon request.
Western blotting. Protein samples were prepared by adding RIPA buffer with protease inhibitor cocktail (Roche) to cells and diluted in SDS-PAGE protein sample buffer. Samples were heated for 5 minutes at 95 °C before fractionation on SDS-polyacrylamide gels. The proteins were then transferred to Immobilon P (Millipore) and incubated with primary antibodies overnight at 4 °C. The membranes were then washed with TBST and incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature. Proteins were visualized with SuperSignal West Dura Extended Duration Substrate or SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).
Xenograft assay. 4-6 weeks old female nude mice were injected subcutaneously of 2 × 10 6 cells resuspended in 100 μl PBS into both hind limbs. Chelerythrine was injected intraperitoneally at a dose of 5 mg/kg at 3-4 days intervals. Tumors were measured by digital caliper and volumes were calculated using the following equation, volume = (width 2 × length)/2. All procedures were carried out in accordance with guidelines by Division of Animal Control and Inspection of the Department of Food and Animal Inspection and Control of Macau and were approved by the Animal Care and Use Committee (ACUC) of the Macau University of Science and Technology.
Dual-Drug combination assay. Breast cancer cell lines were plated in 96-well plates (5,000-40,000 cells/ well), treated with various concentrations of chelerythrine and taxol, either alone or in combination for 72 hours. Cell number was determined by SRB assay. And the combination index (CI) score was calculated using Compusyn software 29 . The effects of drug combination were determined based on the CI values. CI < 0.9, synergy; 0.9 < CI < 1.1, additive effect; CI > 1.1 antagonism 30 .
Statistical analysis. Statistical analysis was performed using Microsoft Excel. Data are shown as mean ± SD and the statistical significance was determined by two-tailed Student's t test. To Table 1, chelerythrine inhibited the growth of TNBC cell lines dose-dependently, with IC50 values of 2.6 μM to 4.2 μM respectively. On the contrary, the tested non-TNBC cell lines were relative resistant to chelerythrine treatment compared to TNBC cell lines. The IC50 values of all four non-TNBC cell lines were greater than 10 μM, and two of the four cell lines, MDA-MB-453 and ZR-75-1, did not show any inhibitory effect even at the highest concentration tested. When treated with 5 μM of chelerythrine, all four TNBC cell lines demonstrated significant reduction of cell growth in a time dependent manner, while the four non-TNBC cell lines showed much less inhibition of cell growth at each time point (Fig. 1B). So TNBC cells lines are more sensitive to chelerythrine treatment compared to non-TNBC cell lines in a dose-and time-dependent manner. The selective anti-proliferative activity of chelerythrine on TNBC cells were further confirmed by colony formation assay (Fig. 1C). Colony formation ability of TNBC cells were dramatically decreased after treatment with chelerythrine for 3 hours and almost no cells grew into colonies after 6 hours, while non-TNBC cells reserved largely of their colony formation ability even after treatment for 48 hours.

Chelerythrine selectively induces apoptosis in TNBC cell lines.
Cell cycle arrest is the key cellular event contributing to reduced proliferation, so we first analyzed the effect of chelerythrine on cell cycle progression. Consistent with the unnoticeable cell growth inhibition of 5 μM chelerythrine on non-TNBC cells ( Fig. 1B and C), the cell cycle distribution of these non-TNBC cells were basically unchanged with chelerythrine treatment (5 μM) for 24 hours (Supplementary Figure 1A Figure 1A and B). So chelerythrine affect cell cycle distribution is cell line dependent, indicating other mechanisms existed for its selectivity on inhibition of TNBC cell growth. It is notable that a substantial proportion of nuclei had a sub-G0/ G1 DNA content characteristic of apoptosis upon chelerythrine treatment in all TNBC cell lines, which implies induction of apoptosis could be the cause of selectively anti-proliferative effect of chelerythrine against TNBC cells.
Chelerythrine has been reported to induce apoptosis in lymphoma cells 31 , osteosarcoma cells 32 , squamous cell carcinoma lines 33 and melanoma cells 34 . So we evaluated whether apoptosis is accounted for the selective Representative colony formation assay plates are shown, which were quantified by counting colony number (n = 4). Data are shown as mean ± SD. P-values determined by Student's t-test compared to control. *P < 0.05; **P < 0.01; ***P < 0.001.
anti-proliferative activity of chelerythrine on TNBC cells. As expected, chelerythrine indeed caused chromatin condensation and nuclear fragmentation, the typical morphological characteristics of apoptosis, in TNBC cells but not in non-TNBC cells as visualized by Hoechst staining (Fig. 2A). This was further evidenced by the detection of the cleaved nuclear poly (ADP-ribose) polymerase (cPARP), a marker of apoptosis, in TNBC cell lines compared with non-TNBC cell lines after treatment with chelerythrine by western blotting (Fig. 2B). We then quantified the apoptotic cell fractions by flow cytometry with annexin V and propidium iodide double staining after 24 hours incubation with chelerythrine. The mean percentages of apoptotic cells after chelerythrine treatment of TNBC cell lines MDA-MB-231, BT-549, HCC1937 and MDA-MB-468 were 67.8%, 51.0%, 22.2% and 35.3% respectively, whereas all four non-TNBC cell lines remained viable under the same treatment (Fig. 2C). The induction of apoptosis with chelerythrine treatment in TNBC cells was also time-dependent as exemplified by MDA-MB-231 cells (Fig. 2D). In short, the distinct apoptosis induction activity of chelerythrine on TNBC cells versus non-TNBC cells contributes to its selective anti-proliferative activity on breast cancer cells.

PKN2, one of PKC isozymes, is highly expressed in TNBC cells.
Chelerythrine is known as a highly specific inhibitor of protein kinase C, a family of serine/threonine protein kinase enzymes consisting of at least 12 members that can be categorized into four subgroups specified by their divergent regulatory domains 35 . All the PKC isozymes share a highly conserved kinase/catalytic domain that chelerythrine can interact with and thus inhibits their activities 36 . So we reasoned that one or more PKC isozymes are highly expressed and required for proliferation in TNBC cells compared to non-TNBC cells. By inhibiting the PKC subtype(s), chelerythrine selectively suppresses the growth of TNBC cells.
Among all 12 isotypes of PKC, PRKCA, is the only isoform that has been associated with TNBC both in cell lines and in patient tumors 22,23 . So we first checked the expression levels of PRKCA in breast cancer cell lines. However, PRKCA was only highly expressed in two of the TNBC cell lines, MDA-MB-231 and BT-549, as manifested by both the mRNA level (Supplementary Figure 2A) and the protein level (Supplementary Figure 2B). Which was consistent with the fact that only in these two cell lines, colony formation was significantly decreased when PRKCA was knocked down by lentivirial-mediated shRNA (Supplementary Figure 2C and D). The data implies that the selective anti-proliferative activity of chelerythrine on TNBC cell lines cannot be fully explained by the expression levels of PRKCA.
Since chelerythrine is a pan PKC inhibitor, we then proposed that other PKC subtypes may also be essential in TNBC cell proliferation. We retrieved gene expression profile data of 12 PKC isozymes in 56 human breast cancer cell lines, including 26 TNBC cell lines and 30 non-TNBC cell lines, from the Cancer Cell Line Encyclopedia (CCLE) database 28 . Besides PRKCA, we identified two other PKC subtypes, PKN1 and PKN2, which were significant higher in TNBC cell lines than in non-TNBC cell lines (Fig. 3A). PKN1 mRNA was significantly higher in TNBC cell lines, however the protein level was not (data not shown). PKN2 mRNA level was higher in all four TNBC cell lines than non-TNBC cells even though it was not significant in MDA-MB-231cells (Fig. 3B). It was confirmed by western blotting analysis of PKN2 proteins in breast cancer cell lines tested in this study (Fig. 3C). It is notable that PKN2 is one of the highest expressed PKC isoforms in breast cancer cell lines (Fig. 3A), implicating the importance in tumorigenesis. So we focused on PKN2 as the main target of chelerythrine in this study.

PKN2 expression is essential for TNBC cell growth.
To test whether PKN2 was required to support the growth of TNBC cells, we stably introduced shRNA targeted against PKN2 (Fig. 4A) and examined colony formation of TNBC cell lines and non-TNBC cell lines. As expected, knocking down PKN2 in TNBC cell lines significantly decreased colony formation (Fig. 4B). In contrast, there were no obvious changes in colony formation in all four non-TNBC cell lines (Fig. 4B).
We next sought to test whether increased PKN2 expression in TNBC cell lines is also necessary for cell growth by xenograft assay. TNBC cell line MDA-MB-231 and non-TNBC cell line ZR-75-1 were first transduced with either nonspecific shRNA or PKN2-specific lentivirus and then were injected into contra-lateral hind limbs of athymic nude mice with equal number of cells. PKN2 deficiency did not change xenograft formation of non-TNBC cell line ZR-75-1 but significantly decreased the growth of TNBC cell line MDA-MB-231 (Fig. 4C).
Both the in vitro colony formation assay and the in vivo xenograft assay after PKN2 knockdown underscored its important roles in supporting TNBC cell growth. So inactivation of PKN2 by chelerythrine selectively inhibits TNBC cell growth.

Chelerythrine inhibits xenograft formation of TNBC cells and enhances chemotherapy activity of taxol.
Chelerythrine is able to selectively inhibit in vitro TNBC cell growth as shown above, highlighting the possible application of the reagent for TNBC tumors as a novel targeted therapy. We next tested its in vivo activity in xenograft tumor study. The xenograft tumor growth rates were similar between TNBC cell line MDA-MB-231 and non-TNBC cell line ZR-75-1 (Fig. 4C). When treated with chelerythrine, the growths of MDA-MB-231 tumors were significantly suppressed compared to ZR-75-1 tumors (Fig. 5A).
Currently there is no targeted therapy available for TNBC tumors and chemotherapy is still the standard regime for TNBC patients. We, therefore, examined the effect of chelerythrine in combination with chemotherapy agent taxol on the proliferation of TNBC cells. Treatment with dual drug combination significantly decreased cell proliferation than either drug given individually in all four TNBC cell lines (Fig. 5B). Furthermore, dual treatment with both chelerythrine and taxol had either additive effect or synergistic effect as manifested by combination index (CI) values at ED50 ranging from 0.75190 to 1.13763 (Table 2). Our data suggest that chelerythrine enhances the anti-proliferative effect of taxol on TNBC cells, and combination of PKC inhibitors with chemotherapy agents may be a promising regime for TNBC treatment. . Data are shown as mean ± SD. P-values determined by Student's t-test compared to control. **P < 0.01; ***P < 0.001.

Discussion
In summary, we have shown that chelerythrine selectively inhibits proliferation of TNBC cell lines both timeand dose-dependently, which was consistent with dramatic decrease of colony formation in TNBC cell lines treated with chelerythrine. We further demonstrated that the selective anti-proliferative activity of chelerythrine on TNBC cells is resulted from substantial induction of apoptosis. Finally, chelerythrine significantly inhibited in vivo xenograft formation and increased the cytotoxic effect of chemotherapy agent taxol against TNBC cell lines. Our data suggest chelerythrine might be a promising regimen selectively targeting TNBC tumors which warranted further study. We found PKN2 is the only PKC isoform highly expressed in all TNBC cell lines compared to non-TNBC cell lines tested in this study ( Fig. 3B and C), and suppression of PKN2 significantly decreased colony formation (Fig. 4B) and xenograft growth (Fig. 4C) in TNBC cells, indicating PKN2 is the target of chelerythrine responsive for its selective activity on TNBC cells. PKN2 plays important roles in cellular processes, including cell cycle progression 37 , actin cytoskeleton assembly 38 , cell adhesion 39 , tumor cell migration and invasion 40 and apoptosis 41,42 .
Our findings indicate that PKN2 is required for TNBC cells growth, which could be a potential diagnostic biomarker or a therapeutic target for TNBC. However, the hypothesis was only tested in limited cell lines in this study. It is needed to be further verified in more breast cancer cell lines and clinical specimens.
We also showed that PRKCA, the alpha isotype of PKC, was overexpressed in two TNBC cell lines tested in this study (Supplementary Figure 2A and B), and knocking down PRKCA suppressed colony formation in the two cell lines (Supplementary Figure 2D). Our data indicates that PRKCA is required for the growth in these two TNBC cell lines, which is consistent with the findings that PRKCA is associated with TNBC both in cell lines and in patient tumors 22,23 . So even though we identified PKN2 as the possible target of cherethrine responsive to the selective activity against TNBC cells, we still cannot rule out other PKC isozymes, like PRKCA, also play roles in the phenomenon observed in this study. Further studies are needed to clarify the functions of each individual PKC isoform in TNBC tumor development. Combined inhibition of PKC isoforms will be a more precise  approach to TNBC tumors based on expression and function of certain PKC isozymes. In fact, there is still a long way to reach the goal, as rare PKC isozyme-specific inhibitors are available 43,44 . However, simultaneously inhibiting all isozymes by pan PKC inhibitors, like chelerythrine, is still worth to try on TNBC tumors. As we showed greater activities of chelerhthrine on cell growth (Fig. 1) and induction of apoptosis (Fig. 2) in TNBC cell lines overexpressing both PKN2 and PRKCA (MDA-MB-231 and BT-549) in general.
We have also shown that chelerythrine selectively induces apoptosis in TNBC cell lines tested in this study. PKN2 is rapidly and specifically cleaved by caspase-3 within its regulatory domain, during the induction of apoptosis 41 . And the cleaved C-terminal peptide of PKN2 binds to Akt and down regulates its kinase activity, resulting in the amplification of pro-apoptotic signalling in the cell 42 . PRKCA exhibits anti-apoptotic function by suppressing p53 dependent activation of IGFBP3 45 or by phosphorylation of Bcl2 46 . It will be interesting to test whether chelerythrine induce apoptosis through regulation of these pathways. On the other hand, chelerythrine has been reported inducing apoptosis by inhibiting anti-apoptotic protein Bcl-2 47 , by dephosphorylation of ERK and Akt 48 , by activation of p53 49 , by inhibiting the expression of cytoprotective proteins HSF1 and hsp70 31 , by inducing reactive oxygen species (ROS) 50,51 , and so on. It will also be interesting to test whether inhibition of PKC, especially PKN2 and PRKCA isoforms, either individually or simultaneously, leading to apoptosis through these pathways.
Most of breast cancer survivors died from noncancer causes, especially for those 50 years and older. And among these causes, cardiovascular disease (CVD) represents the greatest single factor, accounting for approximately 35% of noncancer mortality 52 . CVD caused death is associated with breast cancer treatments, like radiotherapy 53 , HER2 targeted therapy 54 , and chemotherapy 55 . Compared to non-TNBC patients, TNBC patients have a higher prevalence of metabolic syndrome and obesity [56][57][58][59] , both are well known as CVD risk factors. While PKC activation is well documented in CVD developmet 60,61 , and chelerythrine has been reported to improve diabetes induced endothelial dysfunction in rats 62 . All these evidences may add to the value of chelerythrine for TNBC treatment. Which could benefit the patients not only by inhibiting tumor growth, but also by improving CVD complications.