PMA synergistically enhances apicularen A-induced cytotoxicity by disrupting microtubule networks in HeLa cells

Human HeLa cervical cancer cells (ATCC, Rockville, MD) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and antibiotics. Cells were maintained at 37°C, 5% CO₂ and 95% air.

Apicularen A was provided by Dr. Ahn (Division of Ocean Science, Korea Maritime University, Busan, Korea) and dissolved in dimethyl sulfoxide. Phorbol 12-myristate 13-acetate (PMA), thiazolyl blue tetrazolium bromide (MTT), anti-α-tubulin and anti-β-tubulin antibodies were purchased from Sigma (St Louis, MO, USA). Anti-PARP and anti-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-caspase-3 antibody was purchased from R&D Systems (Wiesbaden, Germany). Z-VAD-fmk, Ro31-8220 and Go6983 were purchased from Calbiochem (San Diego, CA, USA). All other reagents were molecular biology grade.

Cell viability was assessed by thiazolyl blue tetrazolium (MTT) assay. Exponentially growing cells were exposed to apicularen A in the presence or absence of PMA for 24 and 48 hours. MTT solution was added to each well (0.5 mg/ml) and incubated for 2 hours. Cell viability was assessed by measuring the absorbance at 570 nm in an ELISA plate reader.

The cells were lysed using buffer containing 300 mM Tris–HCl (pH 7.5), 100 mM NaCl, 10 mM EDTA, 200 mM sucrose and 0.5% SDS. Intracellular DNA was extracted with phenol/chloroform (1:1) and chloroform/isoamylalcohol (24:1). DNA was precipitated and digested in 10 mM Tris–HCl (pH 8.0), 1 mM EDTA and 40 μg/ml RNase A for 1 hour at 37°C. Then, DNA (10 μg) was resolved by electrophoresis in a 1.2% agarose gel supplemented with ethidium bromide (0.2 μg/ml), and DNA fragmentation was examined by ultraviolet transillumination.

Cell extracts were prepared by suspending 2 × 10⁶ HeLa cells in 100 μL TTE buffer [10 mM Tris–HCl (pH 8.0), 0.5% Triton X-100, 10 mM EDTA] on ice for 30 min, and then centrifuging at 15,000 × g for 10 minutes at 4°C. Lysates (30 μg total protein in 10 μl) were mixed with 90 μl assay buffer [20 mM HEPES (pH 7.5), 10% glycerol, 2 mM DTT] containing 40 μM Ac-DEVD-AFC. Caspase-3 activity was measured at 37°C using a spectrofluorometric plate reader (Perkin-Elmer LS-50B., Foster City, CA, USA) in kinetic mode using excitation and emission wavelengths of 400 nm and 505 nm.

HeLa cells were lysed in buffer containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% nonidet P-40, 0.5% deoxycholate, 0.1% SDS and protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany). Cell lysates were subjected to SDS-PAGE and transferred onto nitrocellulose (Pall Life Sciences, Port Washington, NY, USA) or PVDF membranes (Millipore, Woburn, MA, USA). The membranes were first probed with primary antibodies and then with HRP-conjugated secondary antibodies (Calbiochem), and the proteins were detected using the ECL system (Amersham Biosciences Corp., Piscataway, NJ, USA).

HeLa cells exposed to apicularen A in the presence or absence of PMA were washed with phosphate buffered saline (PBS) and fixed in 70% ethanol at −20°C overnight. Before analysis, cells were centrifuged and incubated with propidium iodide (50 μg/ml) supplemented with RNase A (1 mg/ml) for 30 minutes at room temperature. The relative DNA content was measured by flow cytometry using a Becton-Dickinson FACSort and by manual gating using CellQuest software.

The mRNA level of α-tubulin was measured by RT-PCR. Total RNA was isolated using the TRIzol® reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized using AMV reverse transcriptase at 42°C for 1 hour. The mixture was then boiled for 5 minutes to inactivate reverse transcriptase and quickly chilled on ice. The cDNAs were amplified by RT-PCR using the HiPi Plus PCR master mix (Elpis Biotech, Korea). PCR products were separated on 1.2% agarose gels with ethidium bromide (0.2 μg/mL), and amplification products were examined by ultraviolet transillumination.

HeLa cells were seeded onto glass coverslips and were exposed to apicularen A in the presence or absence of PMA. The cells were washed twice with PBS, permeabilized with 0.25% triton X-100 and 0.5% glutaraldehyde for 1 minute at room temperature, and then fixed with 1% glutaraldehyde for 10 minutes before overnight incubation with anti-α-tubulin antibody diluted 1:500. After washing three times in PBS containing 0.1% Tween-20 (PBS/T), cells were incubated for 1 hour with secondary antibody (Alexa Fluor 488 goat anti-mouse IgG diluted 1:400) in the dark. After washing five times, cells were stained with 20 μg/ml propidium iodide and 1 mg/ml RNase A for 20 minutes at room temperature. Microtubules and nuclei were observed using an FV-500 fluorescence microscope (Olympus, Dulles, VA, USA). The fluorescence intensity was quantified using image J software.

Results are expressed as the means ± SE. Statistical significance was assessed using the Student’s t-test and analysis of variance (ANOVA). P < 0.05 was considered to be significant. The combined effect of PMA and apicularen A (combination index) was calculated using the formula %AB/%A × %B, where A and B are the effects of each individual agent and AB is the effect of the combination. When the ratio (combination index) is 1 the effect is considered additive; when the combination index is significantly greater than or less than 1, the effect is considered subadditive (negative synergism) or supraadditive (positive synergism), respectively [23]. Statistical significance value of the combination index was compared with the additive combination index of 1 by one-sided Student’s t-test.

The effect of apicularen A on HeLa cell growth was performed. Apicularen A decreased cell viability in a concentration and time-dependent manner (Figure 1A). In addition, suspended HeLa cells exposed to apicularen A exhibited membrane blebbing, nuclear condensation and shrinkage of the cytoplasm (Figure 1B). To investigate whether these morphological changes were caused by apoptosis, genomic DNA was purified and analyzed for fragmentation. As shown in Figure 1C, apicularen A induced DNA fragmentation at 48 hours. Since caspase-3 plays a crucial role in apoptotic cell death by cleaving poly (ADP-ribose) polymerase (PARP) to suppress the DNA repair pathway [24], we tested the potential involvement of caspase-3 in apicularen A-induced cell death by measuring caspase-3 activity and the PARP cleavage. HeLa cells exposed to apicularen A exhibited a 3-fold increase in caspase-3 activity compared to control cells (Figure 1D). In addition, apicularen A increased the active form of caspase-3 and the cleaved form of PARP (Figure 1E). Taken together, these results indicate that apicularen A induces apoptotic cell death in HeLa cells.

The role of PKC in the mechanism of action of antitumor agents is controversial since PKC synergizes with some agents and antagonizes others [12, 14, 15, 25, 26]. PMA was used to assess the effect of PKC on apicularen A-induced cytotoxicity. PMA has a similar chemical structure to DAG and activates PKCs by interacting with the DAG binding site [27]. HeLa cells were exposed to 100 nM apicularen A and 20 nM PMA; as shown in Figure 2A and Table 1, PMA synergistically increased the cytotoxicity of apicularen A in a time-dependent manner. This finding was supported by time-lapse video microscopy, showing that combination of PMA and apicularen A strongly induced cell death to a greater extent than apicularen A alone (Additional file 1: Movie S1). Next, flow cytometry was used to assess the potential effect of PMA on the cell cycle. Forty percent of apicularen A-treated cells were apoptotic (sub-G₁ peak) at 48 hours, while no apoptosis was detected in control or PMA-treated cells (Figure 2B). In addition, 80% of the cells exposed to the combination of apicularen A and PMA were apoptotic at 48 hours, indicating that PMA increased apicularen A-induced apoptotic cell death. Since apicularen A induced apoptotic cell death through caspase activation (Figure 1E), we investigated whether the cytotoxicity induced by the combination of PMA and apicularen A also depended on caspase activation. The pan-caspase inhibitor Z-VAD-fmk did not block the cytotoxicity induced by the drug combination (Figure 2C), indicating that the synergy is caspase-independent. Since PMA is involved in several PKC-independent cellular processes associated with cell proliferation and differentiation [28‐30], the role of PKC activation on the synergy with apicularen A was tested by exposing cells to PKC inhibitor Ro31-8220 before adding PMA and apicularen A. Ro31-8220 showed no cytotoxicity in HeLa cells at 48 hours, and pretreatment with Ro31-8220 completely blocked the synergistic apoptotic activity of PMA and apicularen A (Figure 2D and 2E). The PKC inhibitor Go6983 also suppressed the effect of PMA on apicularen A-induced cytotoxicity (Figure 2F). These results suggest that PKC activation increases apicularen A-induced apoptotic cell death.

To identify which PKC isotype is involved in the increase in apicularen A-induced cell death observed in the presence of PMA, cells were pretreated with siRNAs specific for individual PKCs and exposed to PMA and apicularen A. Since PMA activates the classical and novel PKC isotypes and Go6983 inhibits PKCα, β, δ, γ and ζ [27, 31], specific siRNAs against PKCα, β and γ were needed; the isotype-specificity of the knockdown was confirmed by transient transfection and Western blotting in HeLa cells (Figure 3A). As shown in Figure 3B, knockdown of PKCα, but not that PKCβ or PKCγ, significantly decreased the apoptosis induced by the combination of PMA and apicularen A. These results demonstrate that PKCα mediates the synergistic effect of PMA on apicularen A-induced cell death in HeLa cells.

We previously reported that the mechanism of apicularen A-induced apoptotic cell death in human HM7 colon cancer cells partially involved a decrease in intracellular tubulin levels [5]. Thus, we investigated whether PMA increases apicularen A-induced apoptotic cell death by further down-regulating tubulin. Apicularen A decreased total α-tubulin protein levels in a time-dependent manner (Figure 4A). At 48 hours, the combination of PMA and apicularen A decreased α-tubulin protein levels to a greater extent than apicularen A alone, while PMA alone had no effect. Similar results were obtained for β-tubulin (Figure 4B). Since apicularen A down-regulates tubulin levels by decreasing tubulin mRNA levels in HM7 cells [5], tubulin mRNA levels were assessed by RT-PCR in cells exposed to PMA and apicularen A. PMA did not affect α-tubulin mRNA levels in apicularen A-treated cells (Figure 4C). Microtubule architecture was assessed by immunofluorescence using anti-α-tubulin antibody and propidium iodide (PI). PMA-treated cells exhibited similar microtubule architecture to control cells. By contrast, apicularen A induced irregular microtubule networks and nuclear localization, and reduced α-tubulin protein levels. In addition, PMA further increased the effect of apicularen A on the microtubule networks and on α-tubulin levels (Figure 4D). Given that PMA increases apicularen A-induced cell death by increasing PKC activity, the possibility that PKC activation might also be responsible for the effect of PMA and apicularen A on tubulin protein levels was considered. HeLa cells were pretreated with Ro31-8220 and then exposed to apicularen A in the presence or absence of PMA. As expected, inhibition of PKC activity by Ro31-8220 partially restored tubulin levels (Figure 4E). Taken together, these results suggest that the potentiation of apicularen A-induced apoptotic cell death by PMA is associated with decreased tubulin protein levels.