Acta Biochim Biophys Sin, 2017, 1–7 doi: 10.1093/abbs/gmx107 Original Article Original Article Effects of high-intensity focused ultrasound on cisplatin-resistant human lung adenocarcinoma in vitro and in vivo Tao Zhang1, Libin Chen2, Shengmin Zhang2, Youfeng Xu2,*, Yabo Fan1, and Lizhong Zhang1 1 Department of Ultrasound, Ningbo No. 9 Hospital, Ningbo 315020, China, and 2Department of Ultrasound, Ningbo First Hospital, Ningbo 315010, China *Correspondence address. Tel/Fax: +86-574-87085004; E-mail: firstname.lastname@example.org Received 7 April 2017; Editorial Decision 27 June 2017 Abstract It is widely accepted that high-intensity focused ultrasound (HIFU) is a minimally invasive treatment option for different tumors, but its roles and the corresponding mechanism in cisplatin (DDP) chemoresistance in lung adenocarcinoma (LA) remain unclear. In this study, we investigated the response of DDP-resistant LA cells to HIFU and its underlying molecular mechanisms using molecular biology techniques. It was found that HIFU exposure inhibited the proliferation of DDP-resistant A549 (A549/DDP) cells through arresting cell cycle at the G1/G0 phase via the Cyclin-dependent pathway and promoting apoptosis in a Bcl-2-dependent manner. Furthermore, the results also showed that HIFU exposure could down-regulate the expressions of MDR1, MRP1, and LRP mRNAs, as well as P-gp, MRP1, and LRP proteins related to drug resistance in A549/DDP cells. In vivo experiments also demonstrated that HIFU could reduce the size and mass of subcutaneously transplanted tumors produced by A549/DDP cells through mediating Cyclin-dependent and Bcl-2-dependent pathways. These results suggested that HIFU treatment could inhibit the proliferation of DDP-resistant lung cancer cells and might be a novel therapeutic method for patients with DDP resistance. Key words: high-intensity focused ultrasound, A549/DDP cells, growth inhibition, chemoresistance Introduction Lung cancer is the most commonly diagnosed tumor and ranks the ﬁrst for cancer-related death globally , of which the lung adenocarcinoma (LA) accounts for more than 30% of all patients with lung cancer . Currently, complete lung resection is the most effective therapy for LA, but it is not suitable for patients diagnosed at advanced stage. Cisplatin [cis-diamminedichloroplatinum (II); Pt(NH3)2Cl2; DDP]-based chemotherapy is based on the formation of DDP–DNA that leads to DNA damage and sequentially activates the apoptosis signaling pathways in cells, which is the ﬁrst-line therapy for patients with advanced LA [3,4]. However, the development of DDP resistance often occurs in clinical practice and is considered as a major obstacle limiting the success of treatment [5,6]. Therefore, there is an urgent clinical need to seek adjuvant therapy which is effective for DDP-resistant LA cells. High-intensity focused ultrasound (HIFU) is an emerging therapeutic technique that uses ultrasonic waves to induce protein degeneration and coagulation necrosis of tumor tissues by efﬁciently targeting tumor entities . In the past decade, HIFU has been shown to treat different solid tumors without harming the surrounding tissues. In pancreatic cancer, several clinical studies have demonstrated that HIFU is a promising method for local tumor control and palliative pain control [8–10]. Moreover, combination of HIFU and gemcitabine has achieved even better therapeutic outcomes in advanced pancreatic cancer patients [11,12]. In breast cancer, HIFU treatment obviously decreased the expressions of proliferating cell nuclear antigen (PCNA), cell adhesion molecule CD44v6 and MMP-9 . In addition, HIFU could enhance chemotherapeutic drug delivery into tumor tissue by permeabilizing cell outer © The Author 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: email@example.com 1 2 membrane . HIFU ablation combined with DDP was found to reduce tumor volume and increase tumor necrosis of cervical cancer and bladder cancer [15,16]. However, the response of chemoresistant cells to insonation usually differs from that of chemosensitive cells, which might be ascribed to multiple mechanisms underlying the enhancement of DDP with ultrasound [17–19]. Recently, a study showed that HIFU could inhibit human LA cell proliferation and induce cell apoptosis . However, the effect of HIFU on DDPresistant lung cancer cells and its corresponding mechanism have not been revealed. In this study, we investigated the response of DDP-resistant LA to HIFU and its underlying molecular mechanisms using molecular biology techniques both in vitro and in vivo, which may help to develop physical approaches that would resensitize the DDP-resistant cancer cells to chemotherapy. The combination of therapeutic HIFU with chemotherapeutic agents might be used as a targeted tool for optimal therapeutic efﬁcacy. Materials and Methods Construction of DDP-resistant LA (A549/DDP) cell line The human parent LA A549 cell line was obtained from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences (Shanghai, China). The DDP-resistant A549 (A549/DDP) cell line was established in our lab by addition of increasing concentrations of DDP to the parent A549 cell culture according to the procedures reported previously . Both cell lines were cultured in RPMI 1640 medium (GIBCO-BRL, Rockville, USA) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in humidiﬁed atmosphere containing 5% CO2. Effects of HIFU on cisplatin chemoresistance Shanghai, China). The number of colonies was observed and counted under the CKX41 microscope (Olympus, Tokyo, Japan). Flow cytometric analysis of cell cycle and apoptosis For cell cycle analysis, cells were washed with ice-cold PBS, ﬁxed with 70% (v/v) ethanol overnight at −20°C and rehydrated in PBS for 10 min. Then cells were stained with PI/RNase and subject to ﬂow cytometric analysis using a FACS instrument (Becton Dickinson, Mountain View, USA) and CellQuest software (Becton Dickinson). For apoptosis analysis, cells were harvested and ﬁxed with 2.5% (v/v) glutaraldehyde for 30 min. The rate of apoptosis was determined using Annexin V-/7-AAD staining by ﬂow cytometry. Quantitative real-time RT-PCR (qRT-PCR) analysis Total RNA was extracted from either control or HIFU group cells using TRIzol reagent (Invitrogen, Carlsbad, USA) and reversely transcribed to cDNA by M-MLV reverse transcriptase (Promega Corp., Madison, USA) according to the manufacturer’s instructions. The qRT-PCR products were detected using SYBR Green PCR core reagents in a BioRad Connet Real-Time PCR platform (Bio-Rad, Hercules, USA). The primer sequences used were as follows: MDR1 (forward: 5′-GGAGCGGTTCTACGA-3′, and reverse: 5′-ACGAT GCCCAGGTGT-3′); LRP (forward: 5′-AGTCAGAAGCCGAG AAAG-3′, and reverse: 5′-CCCAGCCACAGCAAGGT-3′); MRP1 (forward: 5′-GTCGGAACAAGTCGTGCCTG-3′, and reverse: 5′-CAAA GCCTCCACCTCCTCA-3′); and β-actin (forward: 5′-AAGGCTG TGGGCAAGG-3′, and reverse: 5′-TGGAGGAGTGGGTGTCG-3′). β-Actin was used as an internal reference control. The relative quantiﬁcation of these mRNA levels was performed using the 2–ΔΔCt method. HIFU device and exposure Western blot analysis The prototype of a therapeutic HIFU system used in this study was provided by Chongqing Haifu Technology Co., Ltd (Chongqing, China). A total of 1,000,000 A549/DDP cells were placed in the test tube and exposed to HIFU at the frequency of 1.048 MHz and the intensity of 1000 W/cm2 for 9 s , which was deﬁned as HIFU group. The control group (Ctrl) samples were sham-exposed to HIFU in the same fashion. After HIFU treatment, the cells were incubated in a humidiﬁed atmosphere for 6 h, and then underwent cell functional experiments, including cell viability, colony formation, cell cycle, and apoptosis assays. Each group consisted of at least three experiments for the calculation of the mean values. MTT assay was used to determine cell viability. Brieﬂy, cells were seeded into 96-well plates (2.0 × 103 cells/well) and allowed to attach overnight. Then cells were mixed with 0.5 mg/ml MTT solution (Sigma, St Louis, USA). After 4 h of incubation, 150 μl of dimethyl sulfoxide (DMSO) was added into each well to stop the reaction. The absorbance in each well was measured at 595 nm using a microplate reader (BioTek, Vermont, USA). Cells or tumor tissues were lysed with protein extraction reagent RIPA (Beyotime) with a protease inhibitor cocktail (Roche, Basel, Switzerland). The cell lysates were centrifuged at 14,000 g for 15 min to remove the cellular debris, and protein concentrations were determined by the Bradford methods. Approximately 50 μg of proteins were separated by 10% SDS-PAGE and then transferred to PVDF membrane (Amersham, Buckinghamshire, UK). The membranes were blocked with 40 ml skimmed milk for 12 h and incubated with polyclonal antibodies against CDK2 (1:1000, #2546; Cell Signaling), CDK4 (1:1000, 11026-2-AP; Proteintech), Cyclin D1 (1:1000, 601861-1g; Proteintech), Cyclin E (1:500, #21540; SAB), cleaved caspase-3 (1:500, #9661; Cell Signaling), cleaved PARP (1:1000, #9542; Cell Signaling), Bcl-2 (1:1000, #9645; Cell Signaling), Bax (1:1000, #8345; Cell Signaling), P-gp (1:1000, #4587; SAB), LRP (1:1000, #23145; SAB) and MRP1 (1:1000, #9212; SAB) at 4°C overnight. After 4 times of wash (15 min), the membranes were incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1:500,000, 10494-1-AP; Proteintech) for 2 h at room temperature. GAPDH antibody was used as a control. Protein bands were ﬁnally visualized using super ECL detection reagent (Pierce Inc., Rockford, USA). Colony formation assay Animal experiments Cells were seeded in 6-well plates at 500 cells/well after being trypsinized into single cell suspension. After 7 days of culture, the colonies formed were stained with 500 μl crystal violet solution (Beyotime, Six-week-old BALB/c nude mice were purchased from Shanghai Slac Laboratory Animal Co., Ltd (Shanghai, China). A549/DDP cells were exposed to HIFU but control samples were sham-exposed Cell viability assay 3 Effects of HIFU on cisplatin chemoresistance (Ctrl) in the same fashion. HIFU exposure parameters used in this study were 1.048 MHz and 1000 W/cm2 for 9 s. To produce tumor, HIFU-treated A549/DDP cells (1.0 × 107 cells) were injected subcutaneously into the three mice using a 1 ml needle, respectively. Untreated A549/DDP cells (Ctrl) in same number were inoculated into three mice as control. The animals inoculated with cancer cells were raised routinely, with free access to food and water. The size of solid tumor was measured with a caliper every 3 days for 27 days. Mice were sacriﬁced 27 days after treatment. The experiments on mice had been approved by the Ethics Committee at Ningbo First Hospital. Results Establishment of DDP-resistant LA (A549/DDP) cell line To investigate the acquired resistance of LA cells to DDP, we constructed an A549/DDP subline from the parent A549 cells and performed a series of validation experiments. As shown in Fig. 1, parent A549 cells showed lower cell viability than A549/DDP cells under the same concentration of DDP as revealed by MTT assay, suggesting that A549 cells presented higher sensitivity to DDP treatment than A549/ DDP cells. The IC50 value of DDP in parent A549 cells was 18.54 ± 1.74 μg/ml, which was signiﬁcantly lower than that in A549/DDP cells (34.13 ± 2.14 μg/ml; P < 0.01). This result indicated that a stable DDP-resistant cell line (A549/DDP) was successfully established. Statistical analysis Data were presented as the mean ± SD from at least three experiments. The statistical difference between the mean values of two groups was evaluated by Student’s t-test using SPSS software (version 13.0). The signiﬁcance level was set at a P value of less than 0.05. HIFU inhibited the proliferation of DDP-resistant LA (A549/DDP) cells To investigate the roles of HIFU in the A549/DDP cells, we ﬁrst determined the cell viability of A549/DDP cells after HIFU treatment Figure 1. Characterization of DDP-resistant human LA (A549/DDP) cells Cell viability was determined by MTT assay in A549 and A549/DDP cells after treatment with increasing concentrations of DDP for 24 h. The IC50 values of DDP to A549/DDP and A549 cells were calculated, respectively. **P < 0.01. Figure 2. Effect of HIFU on DDP-resistant human LA (A549/DDP) cells (A) Cell viability was determined by MTT assay before and after exposure to HIFU at 0, 24, 48, 72 and 96 h. (B) Representative photographs of single colony and total colonies in plates were shown in control and HIFU-exposed cells by crystal violet staining. (C) The average number of colonies in control and HIFU-exposed cells was shown. Data are expressed as the mean ± SD of three independent experiments. Ctrl represents A549/DDP cells. HIFU represents A549/DDP cells exposed to HIFU. **P < 0.01, ***P < 0.001. 4 by MTT assay. As shown in Fig. 2A, HIFU exposure signiﬁcantly inhibited the cell viability of A549/DDP cells (P < 0.01). Then the effect of HIFU on colony formation ability of A549/DDP cells was evaluated. Compared with the control cells, the number of colonies formed by HIFU-exposed cells was apparently reduced (Fig. 2B,C, P < 0.001). These results indicated that HIFU could signiﬁcantly inhibit the cell viability and capacity of colony formation in A549/ DDP cells. HIFU inhibited the proliferation of DDP-resistant LA (A549/DDP) cells by inducing cell cycle arrest in G0/G1 phase Then, the effects of HIFU on cell cycle of A549/DDP cells were analyzed by ﬂow cytometry (Fig. 3A). Compared with the control A549/DDP cells, the percentage of HIFU-exposed A549/DDP cells in G0/G1 phase of cell cycle was signiﬁcantly increased and the percentage of cells in S phase was notably decreased (Fig. 3B, P < 0.05, P < 0.001). In addition, the expressions of CDK2, CDK4, Cyclin D1, and Cyclin E proteins were signiﬁcantly downregulated in HIFU-exposed A549/DDP cells compared with those in the control A549/DDP cells (Fig. 3C). These results suggested that HIFU might reverse the DDP resistance of A549/DDP cells by partially inducing G0/G1 phase cell arrest. Effects of HIFU on cisplatin chemoresistance HIFU inhibited the proliferation of DDP-resistant LA (A549/DDP) cells by promoting cell apoptosis Next, the apoptosis of the control A549/DDP cells and HIFUexposed A549/DDP cells was determined by ﬂow cytometry (Fig. 4A). The results showed that the early apoptotic rate was signiﬁcantly increased from 4.41% in the control A549/DDP cells to 13.51% in the HIFU-exposed A549/DDP cells. Similarly, the late apoptotic rate was also increased in the HIFU-exposed A549/DDP cells compared with the control A549/DDP cells (Fig. 4B, P < 0.001). Furthermore, HIFU exposure obviously increased the expressions of cleaved caspase-3 and PARP, and decreased the expression of Bcl-2 (Fig. 4C). These results indicated that HIFU inhibited A549/DDP cell proliferation by partially promoting cell apoptosis. HIFU downregulated the expression of drug resistanceassociated molecules in DDP-resistant LA (A549/DDP) cells To gain further insight into the mechanisms related to the effects of HIFU exposure on A549/DDP cells, we investigated the effect of HIFU on the expression of P-gp/MDR1, LRP and MRP1 using qRT-PCR and western blot analysis. As shown in Fig. 5A–C, HIFU could signiﬁcantly suppress the expressions of MDR1, LRP and MRP1 mRNAs in A549/ Figure 3. Effect of HIFU on cell cycle distribution in DDP-resistant human LA (A549/DDP) cells (A) Cell cycle distribution of control and HIFU-exposed cells was analyzed by ﬂow cytometry. (B) Statistical analysis of the percentage of control and HIFU-exposed cells in G0/G1, S, and G2/M phase, respectively. (C) The expression of CDK2, CDK4, Cyclin D1, and Cyclin E in control and HIFU-exposed cells by western blot analysis. Data are expressed as the mean ± SD of three independent experiments. Ctrl represents A549/DDP cells without exposure to HIFU. HIFU represents A549/DDP cells exposed to HIFU. *P < 0.05, ***P < 0.001. Effects of HIFU on cisplatin chemoresistance 5 Figure 4. Effect of HIFU on cell apoptosis in DDP-resistant human LA (A549/DDP) cells (A) Flow cytometric analysis of apoptosis in control and HIFU-exposed cells. (B) Statistical analysis of early apoptosis and late apoptosis in control and HIFU-exposed cells. (C) The expressions of cleaved caspase-3, PARP, and Bcl-2 in control and HIFU-exposed cells by western blot analysis. Ctrl represents A549/DDP cells without exposure to HIFU. HIFU represents A549/DDP cells exposed to HIFU. Data are expressed as the mean ± SD of three independent experiments. ***P < 0.001. Figure 5. Effect of HIFU on drug resistance-associated expression in DDP-resistant human LA (A549/DDP) cells The mRNA and protein expression of P-gp/ MDR1 (A), LRP (B), and MRP1 (C) was determined by qRT-PCR and western blot analysis. β-Actin and GAPDH were used as internal controls. Data are expressed as the mean ± SD of three independent experiments. Ctrl represents A549/DDP cells without exposure to HIFU. HIFU represents A549/DDP cells exposed to HIFU. *P < 0.05 or **P < 0.01. DDP cells at transcription level. Consistently, the expression of P-gp, LRP and MRP1 proteins was obviously decreased in HIFU-exposed A549/DDP cells compared with the control A549/DDP cells. HIFU inhibited tumor growth derived from DDPresistant LA (A549/DDP) cells in vivo To further verify the effect of HIFU on DDP resistance in vivo, we inoculated control A549/DDP cells and HIFU-exposed A549/DDP cells into the nude mice to developed tumor. As shown in Fig. 6A,B, tumor in the HIFU group grew much slower than tumor in the control group. At the end of the study, on Day 27, tumor weight of the HIFU group (0.6 ± 0.16 g) was only 34.5% of that of the control group (1.74 ± 0.06 g) (Fig. 6C). Compared with control group, HIFU treatment signiﬁcantly decreased tumor weight (P < 0.001). To correlate the biological response with the mechanisms identiﬁed in tumor tissues, the expressions of CDK4, Cyclin D1, Bax and MRP1 proteins were determined by western blot analysis. Consistent with the in vitro results, the expression levels of CDK4, Cyclin D1, and MRP1 were downregulated, but Bax was upregulated in HIFU group 6 Effects of HIFU on cisplatin chemoresistance Figure 6. Effect of HIFU on the tumor inducing ability of A549/DDP cells in vivo (A) Representative images of tumors from mice in Ctrl and HIFU groups. (B,C) The volume and weight of tumors from mice in Ctrl and HIFU groups. (D) Western blot analysis of the expressions of CDK4, Cyclin D1, Bax, and MRP1 in Ctrl and HIFU groups. Ctrl means the tumor was produced in mice by injection of A549/DDP cells without exposure to HIFU. HIFU group means the tumor was produced in mice by injection of A549/DDP cells treated with HIFU. *P < 0.05, ***P < 0.001. compared with Ctrl group (Fig. 6D). These results indicated that tumor growth formed from A549/DDP cells in vivo could be obviously inhibited after HIFU treatment. Discussion Recent studies have demonstrated that combined HIFU and chemotherapy can result in better outcomes, including high pain relief and longer survival by improving total drug levels and distributions of bioavailable drug [23–25]. However, the effects and corresponding mechanisms of HIFU in DDP-resistance LA cells have not been clearly identiﬁed. In the current study, we established DDP-resistant A549/DDP cells to explore the effect of HIFU on DDP-resistant LA cells in vitro and tumor growth in vivo. DDP, a DNA damaging agent, has been used as a systematic chemotherapeutic agent for various human tumors including LA. Unfortunately, some factors, including the reduced intracellular drug accumulation, increased anti-apoptotic proteins and decreased proapoptotic proteins are involved in intrinsic or acquired tumor cell resistance to DDP, leading to limited therapeutic efﬁcacy [26–28]. HIFU, as a valuable tool for minimally invasive tumor ablation, has been demonstrated to induce apoptosis by mediating p53 and Bcl-2 family . In addition, HIFU could decrease the multidrug resistance through decreasing the expression of P-gp protein . In our results, HIFU exposure obviously decreased the chemoresistant ability of A549/DDP cells. Further analysis indicated that HIFU could signiﬁcantly inhibit A549/DDP cell proliferation by inducing cell cycle G0/G1 phase arrest and apoptosis. The Cyclins and their partners (CDKs) play a well-established role in the regulation of the cell cycle, of which CDK2-Cyclin E and Cyclin D-CDK4/CDK6 are required for G0/S transition [30,31]. We found that the expressions of CDK2, CDK4, Cyclin D1, and Cyclin E were remarkably decreased by HIFU treatment in A549/DDP cells. Furthermore, HIFU exposure signiﬁcantly enhanced the expressions of pro-apoptotic proteins, cleaved caspase-3 and PARP, and decreased the expression of anti-apoptotic protein Bcl-2. A similar result was also observed in vivo. These results may provide evidence that ultrasound plays an ever-increasing role in the delivery of therapeutic chemotherapeutic agents . In addition, we investigated the effect of HIFU on the expression of resistance-related proteins. We found that HIFU decreased P-gp, MRP1, and LRP expressions at both mRNA and protein levels in vitro and in vivo. Related research suggests that the reversal of cancer drug resistance in clinical trials is mainly ascribed to several factors, such as low binding afﬁnity, the inhibition of metabolism and the excretion of anticancer drugs . Based on these facts, we deduce that HIFU may inhibit DDP-resistant LA cell proliferation possibly through down-regulating the expressions of resistancerelated molecules. However, there are some limitations in this study. For example, there is still lack of evidence that HIFU, as causation, could reverse DDP resistance phenomenon in cancer cells. Despite the suppressive effects of HIFU on DDP-resistant LA cells, difference still exists between laboratory and clinical trials in the use of HIFU to sensitize chemotherapy, as reported by Yu et al. . In summary, our data demonstrated that HIFU exposure could inhibit DDP-resistant LA cell proliferation via regulating G1/S transition and apoptosis, as well as by down-regulating the expressions of drug resistance-associated molecules. To some degree, these ﬁndings will provide an experimental basis for HIFU as an adjuvant therapy in patients with advanced stage lung cancer. References 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. 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