Study on the effect of a triple cancer treatment of propolis, thermal cycling-hyperthermia, and low-intensity ultrasound on PANC-1 cells

Results

Triple treatment greatly inhibits the viability of PANC-1 cells

The cell viability of PANC-1 cells versus the propolis concentration was performed in a gradient manner. When the propolis was less than 0.5%, as shown in Figure 1A, there was no notable inhibition effect on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) results. However, when it exceeded 0.5%, the cell viability dropped significantly. Therefore, a moderate propolis concentration 0.3% was chosen for the following experiments. Next, the physical stimuli of TC-HT and low-intensity US were introduced to affect the viability of PANC-1 cells. In our study, 10-cycles TC-HT and 2.25 MHz US with intensity 0.3 W/cm² and duration 30 minutes were chosen to avoid the thermotoxicity on PANC-1 cells. As shown in Figure 1B, we found that the combination of TC-HT and US was also innocent to PANC-1 cells, but when 0.3% propolis was involved in the triple treatment, the viability of PANC-1 cells was greatly inhibited. It was also noted that the implementation order of TC-HT and US in triple treatment was influential. When TC-HT was performed prior to US (TC-HT + US) in the presence of 0.3% propolis, US helped to further suppress the cell viability of PANC-1 cells significantly down to 17.1%, cutting more than 80% of the viability of the untreated control and thus approaching the in vitro efficacy of chemotherapy drugs. In comparison, the treatment that US was performed prior to TC-HT (US + TC-HT) showed a less inhibition effect (43.1% viability), and hence we adopted the implementation order TC-HT + US as the protocol of the triple treatment in the subsequent experiments. Furthermore, in all double treatments, only 0.3% propolis + TC-HT showed notable inhibition effect (48.9% viability) on PANC-1 cells, which was consistent with our previous results [28]. However, 0.3% propolis + US performed a relatively poor inhibition effect (65.4% viability) on PANC-1 cells, and as a result it was also not included in the following experiments. Figure 1C showed the light microscope images of PANC-1 cells 24 h after each treatment, and the cell morphologies demonstrated an evident inhibition effect on PANC-1 cells after the triple treatment. Moreover, normal cells such as the human skin cells Detroit 551 (Figure 1D) and human pancreatic duct cells H6c7 (Figure 1E) were not significantly affected by the triple treatment as well as all the other treatments. The result indicates that the triple treatment could have a good selective effect on carcinoma cells and normal cells, which makes it safer and more feasible in anticancer treatment.

Figure 1. Viability inhibition effects of propolis on PANC-1, Detroit 551, and H6c7 cells. MTT assay was conducted to determine the viabilities of PANC-1 cells after the treatment of (A) different propolis concentrations and (B) different combinations of physical stimulations. (C) Representative light microscope images of PANC-1 cells after each treatment, scale bar = 100 μm. The viabilities of (D) normal human skin cells Detroit 551 and (E) normal human pancreatic duct cells H6c7 were measured 24 h after each treatment. Data were presented as the mean ± standard deviation in triplicate. ^***P < 0.001, comparison between indicated groups.

Triple treatment increases intracellular ROS levels synergistically

Intracellular ROS is an important regulator of cell death. It has been reported that heat and low-intensity US could elevate the intracellular ROS level [29, 30]. We further investigated whether ROS was increased in response to the triple treatment, so the fluorescent dye dihydroethidium (DHE) was used in this experiment to determine the level of superoxide radical anion (O₂^‧−) in PANC-1 cells after each treatment. As shown in Figure 2A and 2B, it was found that propolis hardly changed the fluorescence signals. Although propolis + TC-HT significantly deformed the fluorescence intensity distribution in an enhanced manner (1.6-fold increase), it did not significantly differ from the enhancement induced by TC-HT alone. In addition, US elevated ROS levels as well, though not as many as TC-HT. Noticeably, the triple treatment showed a significant accumulation of the intracellular ROS (up to a 2.1-fold increase), which was also significantly higher than the TC-HT + 0.3% propolis treatment. The result suggested that, in the triple treatment, US helped to further boost up the generation of ROS in PANC-1 cells, and could result in enhanced cell death rate after the treatment.

Figure 2. The triple treatment raised the generation of intracellular ROS. (A) Intracellular superoxide radical anion (O₂^‧−) levels of PANC-1 cells were determined by flow cytometry with the fluorescent dye DHE. (B) Quantification of the mean DHE fluorescence levels after each treatment. Data were presented as the mean ± standard deviation in triplicate. ^***P < 0.001, comparison between indicated groups.

Triple treatment increases mitochondrial apoptosis in PANC-1 cells

It has been known that the enhanced intracellular ROS levels were positively correlated to mitochondrial apoptosis [31]. In our work, the apoptotic rates of PANC-1 cells after various treatments were analyzed by the flow cytometry with the fluorescence dye Annexin V and propidium iodide (PI) (Figure 3A and 3B). With the aid of US, the triple treatment further caused 55.3% apoptotic rate, which was significantly higher than the 23.5% apoptotic rate caused by the double treatment of propolis and TC-HT. The cell apoptosis results observed here were highly consistent with the results of the accumulated ROS levels in PANC-1 cells after the same treatment, as described in Figure 2. Furthermore, the mitochondrial membrane potential (MMP) was assessed using the lipophilic cationic fluorescent dye 3,3′-dihexyloxacarbocyanine iodide (DiOC₆(3)) fluorescence staining by flow cytometric analysis. As shown in Figure 3C and 3D, the ratio of the cells exhibiting MMP loss was significantly promoted to 23.3% after the double treatment of propolis + TC-HT, and it was further elevated significantly to 34.7% by employing the triple treatment. These results showed that adopting US in the triple treatment could decrease MMP level, and hence caused more mitochondrial dysfunction. The decreased MMP level was an indicator of mitochondrial apoptosis, and since the results of apoptosis assay (Figure 3A and 3B) and MMP assay (Figure 3C and 3D) were quite similar, we believe that the mitochondrial dysfunction was implicated in the apoptosis of PANC-1 cells via the triple treatment.

Figure 3. Triple treatment increased apoptosis on PANC-1 cells via mitochondrial dysfunction. (A) Apoptosis after treatment was analyzed via flow cytometry with Annexin V-FITC/PI double staining, and (B) the apoptotic percentage (Q2 + Q4) was calculated. (C) MMP level after treatment was analyzed via flow cytometry with DiOC₆(3) staining, and (D) the percentage of cells with the loss of MMP (M1) was calculated. Data were presented as the mean ± standard deviation in triplicate. ^**P < 0.01, ^***P < 0.001, comparison between indicated groups.

The apoptosis induced by triple treatment is regulated through MAPK pathway

The activation of apoptotic signalling was examined by western blot analysis. As shown in Figure 4A, we found that the poly (ADP-ribose) polymerase (PARP) cleavage ratio (cleaved PARP/full length PARP), an indicator of caspase-induced apoptosis [32–34], was significantly increased (2.9-fold increase) after propolis + TC-HT treatment on PANC-1 cells. Noticeably, the PARP cleavage was further promoted significantly to a 6.2-fold increase by US in the triple treatment (Figure 4A). Together with the previous flow cytometry results of apoptosis and MMP, it was pointed out that propolis + TC-HT could activate the mitochondrial apoptosis signalling in PANC-1 cells, and US in the triple treatment could further help this cascade to realize a near-chemotherapy level treatment in vitro.

Figure 4. Triple treatment modulated apoptosis via regulating the MAPK family. Representative western blots of the apoptosis-related proteins and the quantification of (A) the PARP cleavage ratio (cleaved PARP/full length PARP), the phosphorylation level of (B) ERK, (C) JNK, and (D) p38. GAPDH was used as loading control. Data were presented as the mean ± standard deviation in triplicate. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, comparison between indicated groups.

Moreover, it was known that the PARP cleavage could be modulated by MMP level, and mitochondrial dysfunction could also be regulated by the excessive intracellular ROS via MAPK family [35]. In MAPK family, the phosphorylated extracellular signal-regulated kinases (p-ERK) level represented the activation of cell survival [36], while the phosphorylated c-Jun N-terminal kinase (p-JNK) and phosphorylated p38 MAPK (p-p38) levels were the indicator of cell death [37, 38]. In this study, it was found that the p-ERK level was suppressed by propolis + TC-HT treatment (0.30-fold decrease), and was further down-regulated when US was introduced in the triple treatment (0.15-fold decrease) (Figure 4B). In addition, the p-JNK and p-p38 levels both exhibited a reverse performance, which were promoted the most in the triple treatment (8.7-fold and 9.2-fold increase, respectively) (Figure 4C and 4D). These results were consistent with the results of ROS and MMP assessments by flow cytometry. Therefore, we speculated that the excess intracellular ROS induced by the triple treatment regulated the activation of the MAPK family and thus caused mitochondrial dysfunction and the cascade of apoptosis.

ROS scavenger attenuates the apoptosis induced by triple treatment

To further confirm that the cell death after the triple treatment was regulated by the generation of intracellular ROS, the ROS scavenger N-acetyl-cysteine (NAC) was applied in the experiment [39]. 5 mM NAC was incubated with PANC-1 cells 1 h prior to the triple treatment. As shown in Figure 5A, the inhibitory effect of the triple treatment was restored by NAC, and NAC itself did not affect the viability of PANC-1 cells. Similar results were also observed in the activation of apoptotic pathway, as shown in Figure 5B. NAC alone did not affect PARP cleavage, but it significantly down-regulated the triple treatment-promoted PARP cleavage (Figure 5B). Therefore, the results supported our speculation that the triple treatment could induce mitochondrial apoptosis of PANC-1 cells via the excessive increment of intracellular ROS.

Figure 5. ROS inhibition blocked the cell death and the activation of apoptosis pathways. (A) Cell viabilities of PANC-1 cells and (B) the quantification of PARP cleavage ratio (cleaved PARP/full length PARP) after the triple treatment with or without 1 h NAC pretreatment. GAPDH was used as loading control. Data were presented as the mean ± standard deviation in triplicate. ^**P < 0.01, ^***P < 0.001, comparison between indicated groups.

Triple treatment can be applied with chemotherapy drug as a novel anticancer treatment

In this study, we have shown that the method TC-HT followed by mild US exposure could further amplify the anticancer effect of propolis. But, the question is whether the TC-HT + US method can be expanded to the existing chemotherapy drugs. Cisplatin, for instance, was a commonly used clinical chemotherapy drug for several kinds of cancers such as lung, ovarian, breast, and brain cancer [40]. Besides, it has also been reported that cisplatin was sensitive to heat [41]. However, the conventional therapeutic dosage of cisplatin could cause severe side effects to the patients [42, 43], and therefore it was important to develop a new method to reduce the effective dose of cisplatin. As a result, we applied the method of TC-HT + US with cisplatin on the PANC-1 cells to investigate the potential of this triple treatment method. A relatively low dose of 1 μM cisplatin and short incubation time 24 h was chosen for the MTT assay independently or in combination with the physical stimulations [44, 45]. After the treatment, the viability of PANC-1 cells was just slightly inhibited by 7.5% by individual cisplatin, and the double treatment of cisplatin + TC-HT also did not alter the viability of PANC-1 cells. The triple treatment, however, promoted the inhibitory effect significantly up to 48.2% (Figure 6A). Moreover, the apoptotic activity was also boosted up by the triple treatment of cisplatin. As shown in Figure 6B, 1 μM cisplatin alone rarely affected PARP cleavage in PANC-1 cells (0.93-fold decrease), and cisplatin + TC-HT treatment significantly elevated PARP cleavage to a 3.20-fold increase. This elevation was further increased with the help of US (5.82-fold increase). Therefore, compared to the conventional results of cisplatin, our method could not only reduce the effective dose but also boost up the anticancer effect of cisplatin.

Figure 6. The method of triple treatment promoted the inhibitory effect of the heat sensitive chemotherapy drug cisplatin. (A) Cell viabilities of PANC-1 cells and (B) the quantification of PARP cleavage ratio (cleaved PARP/full length PARP) 24 h after the treatment of cisplatin, cisplatin + TC-HT, and the triple treatment of cisplatin + TC-HT + US. Cisplatin concentrations in all treatments were 1 μM. Data were presented as the mean ± standard deviation in triplicate. ^**P < 0.01, ^***P < 0.001, comparison between indicated groups.

Materials and Methods

Cell culture and propolis treatment

The human pancreatic cancer cell line PANC-1 and the normal human embryonic skin cell line Detroit 551 were obtained from Bioresource Collection and Research Center (Hsinchu, Taiwan). Normal human pancreatic duct H6c7 cells were purchased from Kerafast, Inc. (Boston, MA, USA). PANC-1 and Detroit 551 cells were cultured respectively in DMEM and EMEM (both from Hyclone, South Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone) and 1% penicillin-streptomycin (Gibco Life Technologies, Grand Island, NY, USA). H6c7 cells were maintained in keratinocyte-serum free medium (Invitrogen, Life Technologies, Grand Island, NY, USA) supplemented with human recombinant epidermal growth factor, bovine pituitary extract (Invitrogen), and 1% penicillin-streptomycin (Gibco Life Technologies). All cells were maintained in a humidified 5% CO₂ incubator at 37°C and subcultured by 0.05% trypsin–0.5 mM EDTA solution (Gibco Life Technologies). Once the confluences reached suitable percentages, cells were plated in 96-well or 35-mm-diameter culture dishes (Thermo Fisher Scientific, Inc., Waltham, MA, USA) for in vitro experiments after 24 h incubation in the humidified 5% CO₂ incubator at 37°C. Propolis was purchased from Grandhealth™ (Grand Health Inc., Richmond, British Columbia, Canada), and cisplatin was obtained from Sigma-Aldrich (St. Louis, MO, USA). All agents were mixed with culture medium to the desired concentration and were incubated with cells for 1 h before treating physical stimuli.

Ultrasound exposure

The US exposure system consisted of a function generator (SG382; Stanford Research Systems, Sunnyvale, CA, USA), a power amplifier (25a250a; Amplifier Research, Souderton, PA, USA), and a planar transducer (A104S-RM; Olympus NDT Inc., Waltham, MA, USA). Continuous pulses were produced using the function generator with the following parameters: -10 dBm amplitude, 1 ms pulse period, and 0.5 ms pulse width. The cell culture plate or dish was placed on the ceramic transducer (resonance frequency 2.25 MHz), which converted electrical signals into acoustic power (Figure 7A). To avoid undesirable thermal effects induced by US, the output power of the spatial average intensity of the US exposure was adjusted to be 0.3 W/cm² according to the previous studies [64, 65].

Figure 7. Experimental setups for the triple treatment. 35-mm culture dishes were placed on (A) a ceramic transducer and (B) a modified PCR machine for the exposures of US and TC-HT, respectively. (C) The schematic representation of the TC-HT temperature settings. (D) Cell temperature detected by a needle thermocouple when TC-HT was implemented. (E) Experimental schedule of the triple treatment with different exposing order of US and TC-HT.

Thermal cycling-hyperthermia (TC-HT) treatment

A modified polymerase chain reaction (PCR) system was used to perform TC-HT (Figure 7B). Thermal cycler (model 2720) was purchased from Applied Biosystems (Thermo Fisher Scientific). The system was repeatedly brought to the desired high temperature state and followed by a cooling stage to achieve a series of short period of heat exposure within the desired time (Figure 7C). The experimental setup and administration of TC-HT (10-cycles) have been previously described with optimum results [28]. The actual temperatures the cancer cells sensed were measured by a needle thermocouple, ranging in 45~40.5°C (Figure 7D). Ultrasound exposure was applied either before or after the TC-HT treatment, for different combination tests (Figure 7E). During the TC-HT treatment (~45 min), the control and propolis-treated groups were under room temperature (RT) without a 5% CO₂ environment. No significant impact on cell viability of untreated control cells was observed, regardless of whether the cells were moved outside or placed inside the 5% CO₂ incubator during TC-HT treatment (Supplementary Figure 1). After the treatments, cells were maintained in the cell culture incubator for the following experiments.

MTT assay

MTT (Sigma-Aldrich) was dissolved in distilled water to prepare a 5 mg/mL stock solution. The treated PANC-1 cells were incubated for 4 h in the humidified 5% CO₂ incubator at 37°C with a final MTT concentration 0.5 mg/mL in DMEM culture medium to assess the cell viabilities. The formazan crystals were dissolved by equal volume of the solubilizing buffer of 10% sodium dodecyl sulfate (SDS) (Bioshop Canada Inc., Burlington, Ontario, Canada) solution in 0.01 N hydrochloric acid (HCl) (Echo Chemical Co. Ltd., Miaoli, Taiwan) in the humidified 5% CO₂ incubator at 37°C overnight. The absorbance of each well was detected by Multiskan GO microplate Spectrophotometer (Thermo Fisher Scientific), and the quantity of formazan was determined by the absorbance at 570 nm, with a background subtraction at 690 nm. The cell viabilities were expressed in percentage and the untreated control was set at 100%.

Treatment with ROS scavenger

PANC-1 cells were seeded into 96-well or 35-mm-diameter culture dishes overnight. For ROS inhibition analysis, cells were pretreated with 5 mM NAC (Sigma-Aldrich) in culture medium for 1 h in the humidified 5% CO₂ incubator at 37°C, and subsequently treated with propolis and/or physical stimuli.

Apoptotic analysis by flow cytometry

PANC-1 cells were collected 24 h after treatments and then rinsed twice with ice-cold phosphate buffered saline (PBS) (Hyclone). The apoptotic rates were analyzed by the Annexin V-FITC and PI double detection kit (BD Biosciences, San Jose, CA, USA), and the rinsed cells were resuspended in binding buffer containing Annexin V-FITC and PI and then incubated at RT for 15 min in the dark. Apoptotic signals were detected by FACSCanto II system (BD Biosciences).

ROS and mitochondrial membrane potential (MMP) analyses by flow cytometry

ROS was detected using DHE (Sigma-Aldrich), and the loss of MMP was determined using DiOC₆(3) (Enzo Life Sciences, Inc., Plymouth Meeting, PA, USA). PANC-1 cells were harvested 24 h after treatments and rinsed with PBS before staining. Rinsed cells were resuspended and then incubated with 5 μM DHE or 20 nM DiOC₆(3) in PBS at 37°C for 30 min in the dark. The fluorescence signals were measured by FACSCanto II system (BD Biosciences) with the PE channel (for DHE staining) or FL1 channel (for DiOC₆(3) staining).

Western blot analysis

Protein expression levels of PANC-1 cells were quantified by western blot analysis. Cells were rinsed with PBS and then lysed in the lysis buffer (50 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.25% deoxycholic acid, 1% NP-40, 1% Triton X-100, 0.1 % SDS, 1 mM EDTA) (Millipore, Billerica, MA, USA), supplemented with active protease (Millipore) and phosphatase inhibitor cocktail (Cell signaling Technology, Danvers, MA, USA). After centrifugation, the supernatants were collected and the protein concentrations were quantified by Bradford protein assay (Bioshop, Inc.). Equal amount of proteins (20 μg) was resolved by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). 5% skim milk powder or 5% bovine serum albumin in TBST (20 mM Tris-base, pH 7.6, 0.15 M NaCl, 0.1% Tween 20) was used to block nonspecific antibody binding sites for 1 h at RT. Afterwards, the blocked membranes were probed with specific primary antibodies against p-ERK, p-JNK, PARP (Cell signaling), p-p38, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Gentex, Irvine, CA, USA) at 4°C overnight. The membranes were rinsed with TBST buffer three times and then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) in a blocking solution at RT for 1 h. Immunoreactivity signal was amplified by an enhanced chemiluminescence (ECL) substrate (Advansta, San Jose, CA, USA) and detected by an imaging system Amersham Imager 600 (GE Healthcare Life Sciences, Chicago, IL, USA). GAPDH was used as the loading control to normalize the relative folds of targeting proteins.

Statistical analysis

Experiments were repeated three times for validation, and statistical analyses were performed using one-way analysis of variance (ANOVA) by OriginPro 2015 software (OriginLab). Results were expressed as the mean ± standard deviation, and were considered to be statistically significant when P-values were less than 0.05.

Abbreviations

ANOVA: analysis of variance; DHE: dihydroethidium; DiOC₆(3): 3,3′-dihexyloxacarbocyanine iodide; ECL: enhanced chemiluminescence; FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HCl: hydrochloric acid; HIFU: high-intensity focused ultrasound; MAPK: mitogen-activated protein kinase; MMP: mitochondrial membrane potential; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NAC: N-acetyl-cysteine; O₂^‧−: superoxide radical anion; p-ERK: phosphorylated extracellular signal-regulated kinases; p-JNK: phosphorylated c-Jun N-terminal kinase; p-p38: phosphorylated p38 MAPK; PARP: poly (ADP-ribose) polymerase; PBS: phosphate buffered saline; PCR: polymerase chain reaction; PI: propidium iodide; PVDF: polyvinylidene fluoride; ROS: reactive oxygen species; SDS: sodium dodecyl sulfate; SDS-PAGE: SDS-polyacrylamide gel electrophoresis; TC-HT: thermal cycling-hyperthermia; US: ultrasound.

Author Contributions

C.Y.C. conceived and supervised the study. Y.Y.K., W.T.C. and C.Y.C. designed the study. Y.Y.K. performed experiments and collected data. All authors contributed to data analysis. Y.Y.K. and C.Y.C. wrote the manuscript. All authors contributed to the article and approved the final manuscript.

Conflicts of Interest

The authors declare no conflicts of interest related to this study.

Funding

This work was supported by grants from Ministry of Science and Technology (MOST 110-2112-M-002-004, MOST 109-2112-M-002-004, and MOST 108-2112-M-002-016 to C.Y.C.) and Ministry of Education (MOE 106R880708 to C.Y.C.) of the Republic of China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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