Phytotherapeutics oridonin and ponicidin show additive effects combined with irradiation in pancreatic cancer in vitro

The pancreatic cancer cell lines AsPC-1, BxPC-3 and MIA PaCa-2 were cultured in RPMI-1640 medium, the cell line Panc-1 in Dulbecco’s modified Eagle’s Medium (DMEM). Media included 10% FBS and 1% Penicillin/Streptomycin. The cells were cultured at 37°C in 5% CO₂. AsPC-1 and Panc-1 cell lines were purchased from CLS cell line service GmbH, Germany and BxPC-3 as well as MIA PaCa-2 cells from LGC Standards GmbH (ATCC), Germany. Oridonin was obtained from Sigma-Aldrich Corporation, USA and ponicidin from Shanghai Zhanshu Chemical Technology Co., Ltd., China. Both reagents were dissolved in DMSO and stored at −80°C.

Cells were X-irradiated (X-RAD 320, Precision X-Ray, Inc., USA) operated at 320 kV and 12.5 mA. The average dose rate was 1.1 Gy/min. The used filter consisted of 1.5 mm Al, 0.25 mm Cu and 0.75 mm Sn. The tissue culture flasks, in which the cells were cultured, were placed on a PMMA-plate of 1 cm height.

To assess long-term cell survival, a defined number of cells were seeded in 25 cm² tissue culture flasks and incubated for 24 hours. Cell counts were adjusted for the different treatment schemes. Cell lines AsPC-1, BxPC-3 and Panc-1 were treated with oridonin, ponicidin or DMSO (control) for 24 hours as indicated and irradiated afterwards once with 2 Gy or 6 Gy. The dose of each substance and the intensity of irradiation were established through pre-experiments to achieve a survival rate of 20-70% after single treatment alone. MIA PaCa-2 cells were pretreated with lower doses of oridonin and ponicidin due to higher sensitivity to the reagents. 8-9 days (time differs among cell lines) after the treatment cells were fixed in 70% ethanol and stained with 0.2% methylenblue. Colonies, defined as containing at least 50 cells, were counted manually under a light microscope and the plating efficiency, defined as the quotient of counted colonies to plated cells, was determined. The surviving fraction was defined as the ratio of each experiment’s plating efficiency to its control. Cells were plated in triplets to compensate for pipetting errors and at least three independent experiments were performed.

To analyze combined treatments, surviving fractions were normalized to the values of corresponding single reagent treatment using a ratio of the combined treatment’s plating efficiency to the single reagent treatment one’s. Dose-response curves were created using the linear-quadratic model.20

Combination effects were analyzed defining additivity according to Steel and Peckham.21 For this purpose a dose-response curve was calculated that represents the course of the measured control curve at a pre-effect similar to the single reagent treatment’s effect („theoretical control-curve“). Additive effects were defined as area between the measured control curve and the theoretical one. Supraadditive effects were defined as beneath the theoretical control curve and radioprotective effects as above the measured control curve. Independent toxicity was defined as identical curve to the measured control curve (for further information see Suppl. Figure 1).

Since impact on cell cycle distribution was regressing 24 hours after pretreatment a shorter exposition time to oridonin and ponicidin compared to the other experiments was chosen. Simultaneously the dosage was augmented to compensate for shortage in exposition time. Cell lines Panc-1 and MIA PaCa-2 were pretreated for two hours with oridonin, ponicidin or DMSO (control) and irradiated as indicated. To induce a less lethal damage than one would expect of 6 Gy, an irradiation dose of 4 Gy was chosen. Cells were then fixed in ice-cold ethanol and RNA-strands were cleaved by RNase A (AppliChem GmbH, Germany) at the following time points: beginning of the treatment, before irradiation, 12 and 24 hours after irradiation. Then cells were stained with propidium iodide (Sigma-Aldrich Corporation, USA) and the amount of propidium iodide per cell was quantified in a flow cytometer (FACScan, Becton Dickinson and Company, USA). At least 10⁴ ungated cells were analyzed by BD CellQuest Pro 4.0.2 (Becton Dickinson and Company, USA) and the different phases of the cell cycle were determined using the software ModFit LT 3.0 (Verity Software House, Inc., USA). At least two independent experiments were performed.

Since clonogenic assays revealed different sensitivity between AsPC-1, BxPC-3 and Panc-1 cells on the one hand and MIA PaCa-2 cells on the other hand further experiments were done with the cell lines Panc-1 and MIA PaCa-2. To quantify the induction of DNA double-strand breaks Panc-1 and MIA PaCa-2 cells were pretreated with oridonin, ponicidin or DMSO (control) for 24 hours and were irradiated once as indicated. At different time points after irradiation MIA PaCa-2 and Panc-1 cells were fixed using 1% PFA and ice-cold ethanol. Cells were then stained with a γH2AX-antibody (art. no. 16-202A, Merck KGaA, Darmstadt, Germany) and pan-nuclear intensity was measured using a flow cytometer. Results were normalized by creating a ratio of the measured intensity to the intensity of the control probes. At least three independent experiments were performed.

Cell lines Panc-1 and MIA PaCa-2 were treated with oridonin, ponicidin or DMSO (control) for 24 hours. Then lysates of the cells were gained using lysis buffer (10 mM HEPES pH 7.4, 5 mM CHAPS, 5 mM DTT) and inhibitors (PMSF, Pepstatin, Leupeptin, Aprotinin). The protein concentration was determined in a Bradford protein assay and 20 μg to 30 μg Protein were used for Western Blots. A SDS-polyacrylamide-gel-electrophoresis was run using 12% polyacrylamide-gels with a voltage of 160 V and amperage of 250 mA. A wet transfer was performed (80 V, 60 mA) to transfer the proteins on a polyvinylidenfluoride membrane. The membrane was blocked using Roti-Block (Carl Roth. GmbH & Co. KG, Germany). b-Actin (art. no. 4967), GAPDH (art. no. 2118), Ku70 (art. no. 4588) and Ku80 (art. no 2753) primary antibodies were purchased from Cell signaling Technology Inc., USA. XRCC4 (art. no. sc-271087) and secondary antibodies (art. no. sc-2380, sc-2379) were obtained from Santa Cruz Biotechnology, Inc., USA. Dilution was done according to manufacturer’s instructions. X-Ray films were scanned and the density of the protein bands were determined with the software Image J 1.47V (National Institute of Health, USA). Density was normalized by creating a ratio of protein band to house-keeping protein’s one to avoid minimal differences in the amount of protein load. Then another ratio of the treatment’s value to its control was calculated and allowed to quantify densities and treatment differences. At least two independent experiments were performed.

The results of each experiment were generated as described above. Statistical analysis of the results was done with an unpaired two-tailed t-test using Microsoft® Excel® 2008 for Mac 12.3.6 (Microsoft Corporation, USA). The dose control curves and the figures were calculated and generated with SigmaPlot 12.0 (Systat Software Inc., USA). Results are presented as mean values +/− standard deviation. P < 0.05 was considered as a significant difference between the compared results. *P < 0.05, **P < 0.005, ***P < 0.001.

To investigate long-term survival cells were treated with oridonin or ponicidin in a low dosage of 0.5 μg/ml and high dosage of 2.5 μg/ml (Figure 1A). Since MIA PaCa-2 cells exposed a higher sensitivity to both oridonin and ponicidin, we used 0.1 μg/ml and 0.5 μg/ml as the low and the high dosage in these cells, respectively. MIA PaCa-2 cells showed a significant reduction in clonogenic survival to a fraction of 51% when treated with the lower dosage of oridonin and thus were much more sensitive than the other cell lines, with a mean surviving fraction of 88% in AsPC-1, BxPC-3 and Panc-1 cells (Suppl. Figure 2). Interestingly, ponicidin was more effective than oridonin: 2.5 μg/ml of ponicidin lead to a mean surviving fraction of 12%. The same amount of oridonin showed an almost threefold higher surviving fraction of 32% in AsPC-1-, BxPC-3- and Panc-1 cells (Suppl. Figure 3).

Figure 1

A further dose-dependent reduction in survival could be observed by combination with irradiation. The mean surviving fraction of 88% after single treatment with low dosage of oridonin could be further attenuated by additional irradiation with 2 Gy or 6 Gy to mean 63% and 13%, respectively.

Using these results we investigated the radiosensitivity according to the model of Steel and Peckham as described in the methods section.21 The higher dose of oridonin and ponicidin lead to additive effects in almost all investigated cell lines when combined with 2 Gy or 6 Gy irradiation, whereas the lower dose just showed independent toxicity (Figure 1B and Suppl. Figure 4). Radioprotectivity as well as supraadditivity could not be observed.

To determine changes in cell cycle, the four pancreatic cancer cell lines were treated with oridonin or ponicidin alone as well as in combination with irradiation. Cell cycle phases were analyzed 12 and 24 hours after irradiation. Pretreatment with oridonin (Figure 2A) or ponicidin (Figure 2B) induced a clear G2/M-arrest of the cell lines AsPC-1 and Panc-1 after 12 hours with up to 36% (Figure 2 and Suppl. Figure 5). The G2/M-fraction of combined treatments was not as high as the one observed after irradiation alone but was augmented in time leading to the fact that 24 hours after irradiation there still was a clear G2/M-arrest in the cell lines AsPC-1, BcPC-3 and Panc-1 when treated with the combined therapy. MIA PaCa-2 cells showed a clear G2/M-arrest 24 hours after combined treatment with ponicidin but not with oridonin (Suppl. Figure 5). We did not observe a sub-G1 fraction as surrogate of apoptosis.

Figure 2

The potential influence of oridonin or ponicidin on the DNA double-strand break repair was analyzed by measuring the pan-nuclear γH2AX-intensity of pretreated cells via flow cytometry. Both oridonin (Figure 3A) and poncidin (Figure 3B) lead to an approximately 2.5-fold increase of the γH2AX-intensity shortly after treatment, independent of the particular dose level (7.5 μg/mL vs. 15 μg/mL). There was an increase of γH2AX after irradiation alone but highest γH2AX-intensities could be observed in combined treatments. The Intensity of γH2AX already declined 10 hours posttreatment. We could not observe a difference in the relative reduction of the γH2AX-intensity between irradiation alone and combined treatments.

Figure 3

To determine the influence of oridoinin or ponicidin on the repair of DNA double-strand breaks, Panc-1and MIA PaCa-2-cells were treated with either agent and three main proteins of the NHEJ pathway, namely XRCC4, Ku70 and Ku80 were detected by Western Blots. Pretreatment with oridonin or ponicidin lead to a slight reduction of XRCC4-expression but there was no considerable change in Ku70 and Ku80 protein expression in MIA PaCa-2 cells. Panc-1 cells showed no stringent effect in XRCC4 protein expression but a dose-dependent increase of Ku70 and Ku80 after pretreatment with oridonin or ponicidin (Figure 4).

Figure 4