Research ArticleResearch Article

Experimental Study of Ultrasound on MDR in a Human Tumor in Vivo

Baojin Zhai, Liming Chen, Yumian Guo, ZhiHong Wang and Feng Wu
Chinese Journal of Clinical Oncology December 2007, 4 (6) 390-396; DOI: https://doi.org/10.1007/s11805-007-0390-3

Abstract

OBJECTIVE To evaluate the potential efficacy of low-intensity ultrasound (US) in combination with anticancer drugs to reverse multidrug resistance (MDR) in nude mice.

METHODS A total of 40 male and female athymic nude mice were inoculated subcutaneously with 5×106 HepG2/ADM and HepG2 cells. Ultrasound with pulsed irradiation at an average intensity of 0.5 W/cm2 was given to the tumor area 10 min after administration of adriamycin (ADM). The tumor 3 dimensional diameters were measured by calipers before and after treatment, and the tumor growth indexes (TGI) calculated. RT-PCR was used to detect the gene levels of the HepG2/ADM cells. Immunohistochemical analyses for MDR proteins were conducted on the tumor tissues.

RESULTS The ultrasonic treatment resulted in an average reduction in the tumor volume of 62% one month later. The relative mRNA levels of MDR1 and MRP were significantly different among the following 4 groups: untreated group as control, ADM treated; US treated; and ADM plus US treated. The mRNA levels of mdr1 and mrp were down-regulated in the US groups compared to those of the non-ultrasound groups by multiple comparisons. The relative mRNA levels of lrp expression were not significantly changed. The results of immunohistochemistry indicated that tumor tissue from animals treated with US had remarkably low mdr1 and mrp expression.

CONCLUSION The results showed that low-intensity US can effectively reduce the size of adriamycin-resistant human hepotacarcinoma in a nude mouse model, and support the efficacy of US to overcome multiple mechanisms of drug resistance.

KEYWORDS:

keywords

INTRODUCTION

It is widely accepted that drug resistance is a major obstacle to cancer chemotherapy. Cross-resistance of cancer cells to many types of drugs which show little structural similarity is termed multidrug resistance (MDR)[1]. Although the etiology of MDR is multifactorial, the overexpression of P-glycoprotein (P-gp), a membrane protein that mediates the transport of MDR drugs, remains the most common alteration underlying MDR in laboratory models[2]. Moreover, expression of P-gp has been linked to the development of MDR in human cancers, particularly in leukemias, lymphomas, multiple myeloma, neuroblastoma, and soft tissue sarcoma[3,4]. Recent studies have shown that tumor cells expressing MDR-associated protein (MRP)[5] and lung resistance protein (LRP)[6] also may cause MDR. In an effort to restore cytotoxicity to many of these anticancer drugs, many agents have been studied for their role in reversing MDR. But these agents have been found to be weak inhibitors that were toxic at high doses. Therefore, methods of overcoming MDR are urgently needed.

Since the focus of ultrasonic waves can be localized and their intensity level is relatively easy to control, the use of ultrasound (US) for enhanced cancer therapy has been the subject of much cancer research[7]. Earlier studies have demonstrated that ultrasonic cavitation may cause irreversible cell damage and modify the membrane structure and functional properties of cells[8,9]. In fact, morphological changes on cell surface membranes were detected after the cells were exposed to diagnostic US[10]. Malignant cells were found to be sensitive to ultrasonic treatment, resulting in a transient decrease in cell proliferation[11]. Subsequent to these findings, we have demonstrated that low-intensity US may reverse MDR of the human hepotacarcinoma cell line (HepG2/ADM)[12].

The aim of this study was to further study the use of low intensity US with anticancer drug treatment of adrimycin-resistant solid tumors.

MATERIALS AND METHODS

Animal model production

The ability of US to potentiate the anti-tumor activity of ADM was evaluated using HepG2/ADM xenografts. The resistant (HepG2/ADM) human hepatocarcinoma xenografts were produced as follows: a HepG2 and HepG2/ADM cell suspension was centrifuged, the supernatant removed and adjusted to 5×107 cells/ml. Forty nude mice were injected subcutaneously with 0.9 ml of the HepG2/ADM cell suspension into the scapular area. These transplanted nude mice, were randomly divided into 4 groups with 10 mice per group: ① Control group (HepG2/ADM/nude, no treatment); ②ADM group, ADM (5 mg/kg) was administered via the tail vein (HepG2/ADM/nude+ADM); ③US irradiation group (HepG2/ADM/nude+US); and ④ADM+US group(HepG2/ADM/nude+ADM+US). The 3-dimensional diameters of the tumors were measured by a caliper before treatment.

When the tumors had reached a mean diameter of 0.5 cm, the HepG2/ADM xenografts exhibited high levels of P-gp and mrp mediated drug resistance (Fig.1). The 4 groups of mice were treated with various regimens on day 0 (start of treatment), 7, 14, 21, 28, 35, 42 and 49 with US and i.v ADM (5 mg/kg, obtained from Sigma). Control animals received no treatment. In the US+ADM group, the ADM was administered 10 min before US irradiation. Administration of all of the i.v. compounds was via a tail vein. This study was conducted in accordance with the guidelines of the National Institutes of Health Guide for the Care and Use of Animals after review of the protocol by the Institutional Care and Use Committee. The mice were euthanized when tumors reached 10% of their total body weight.

Fig.1.
Fig.1.

Target and β-actin PCR products of mdr1, mrp and Irp. Total RNA (0.15 mg) extracted from parental (HepG2, lane 1) and resistant sublines(HepG2/ADM, lane 2) xenografts(HepG2/nude and HepG2/ADM/nude; lanes 3 and 4). The products were resolved on a 1.5% agarose gel and stained with ethidium bromide. Lane M represents a 100-bp DNA ladder as a size marker.

Apparatus and experimental method

The ultrasonic apparatus (HIFU Co., Ltd., Chongqing, China) used, produced low-intensity ultrasound, with a resonant frequency of 0.8 MHz. The transducer employed had a diameter of 3.0 cm which was horizontally directed to impact the tumor tissue through a gel. The US transducer made contact with the skin over the tumor and was manually scanned at a rate of 1 mm/s. Ultrasound exposure (0.5 W/cm2 at 0.8 MHz) was applied for a period of 5 min, the pulse ratio was defined as (1:8). The temperature was measured hypodermically by a thermocouple in the xenografts within the power and time range used. The temperature was maintained at 41°C during the treatment, and only an insignificant change (less than 1°C) was observed.

Tumor growth inhibition studies

Tumors appeared at the graft site 2~5 weeks after implantation. Mice bearing growing tumors with a volume of 60~100 mm3 were individually identified and randomly assigned to the control or a treatment group, and the treatment started. Volumes of individual tumors were calculated every seven days from the measurements of two perpendicular diameters using a caliper. Each tumor volume (V) was calculated according to the following formula[13]: V=a2×b/2, where a and b were the smallest and largest perpendicular tumor diameters. RTVs (relative tumor volume) were calculated from the formula: RTV=(Vx/V1), where Vx= the volume on day x, and V1=the tumor volume at the initiation of therapy (day1). Growth curves were obtained for each individual tumor by plotting values of RTV against time (expressed as days after the start of treatment). The inhibitory activity was evaluated according to three criteria: ①the tumor growth inhibition, which was calculated according to the following formula: 100-(RTVt/RTVc)×100 where RTVt is the mean RTV of the treated group and RTVc is the mean RTV of the control group at the time of optimal response; ②the tumor growth delay, calculated as the time in days required for the tumors to reach a 15-fold increase in RTV, corresponding to the survival time of treated mice; prolongation of survival was calculated as the ratio between the survival time in the treated group compared to the controls; ③ the tumor doubling time was calculated as the delay in days required to double an initial tumor volume of 200 mm3 (size in exponential growth phase).

PCR for MDR-1, MRP and LRP mRNA expression

Total RNA of the tumors was extracted with TRIZOL reagent (Life Technologies, Inc.), and the RNA was kept snap-frozen in liquid nitrogen and stored until use. mdr-1, MRP and LRP gene expression was analyzed by reverse transcripase-PCR[14]. Levels of human-specific action were measured as an endogenous control for cDNA synthesis. The primers listed below were selected for their specificity and selectivity for human gene sequences and purchased from Oligo Express (TaKaRa Biotechnology Co., Ltd.): ①mdr1-1, antisense, 5’-ATA TGT TCA AAC TTC TGC TCC TGA-3’; ②mdr1-2, sense, 5’-TGT ACC CAT CAT TGC AAT AGC AGG-3’; ③mrp-1, antisense, 5’-GTA CAC GGA AAG CTT GAC-3’; ④mrp-2, sense, 5’-GGT CAC GCA CAG CAT G-3’; ⑤lrp-1, antisense, 5’-TCT GAG CAT GGC CGT GGA GA-3’; ⑥lrp-2, sense, 5’-TGG AGC CAT CGG TGA TGA GG-3’; ⑦β-actin-1, antisense, 5’-GAC AAG TCT GAA TGC TCC AC-3’; ⑧β-actin-2, sense, 5’-TAT CCA GCG TAC TCC AAA GA-3’.

An aliquot of each reaction mixture was then analyzed by electrophoresis on 1.5 % agarose gel. Densitometry was performed using a UVP gel image analysis system (BIO-RAD, USA) and the ratio between the target and control PCR products was determined by dividing the densitometric volume of the target band by that of the control band.

PCR conditions

The final PCR reaction volume was 50 μl. The cDNA solution (2.5 ml) was pipetted into a sterile 0.2 ml tube, and the following mixture containing 5 ml of 10×Taq buffer (Promega, Madison, WI), 1 ml of dNTP (final concentration, 2.5 mm each), 1 ml of each of the 5’ and 3’ primers (100 ng/ml), 38.5 ml of water, and 0.5 ml (2.5 units) of Taq polymerase (Appligene-Oncor) was added. After preheating at 94°C (hot start), the tubes were placed in a Perkin-Elmer 2400 thermocycler (Yvelines, France) for 5 min at 94°C followed by: (a) for the mdr-1 and lrp gene, 2 cycles (1 min at 94°C, 1 min at 62°C, and 1 min at 72°C) and 38 cycles (1 min at 94°C, 1 min at 60°C, and 1 min at 72°C), and a final elongation at 72°C for 10 min; (b) for the MRP gene, 35 cycles (1 min at 95°C, 1 min at 52°C, and 1.5 min at 72°C) and a final elongation at 72°C for 10 min; and (c) for the β microglobulin gene, 3 min at 94°C, 35 cycles (1 min at 94°C, 30 s at 60°C, and 30 s at 72°C) and 10 min at 72°C.

Statistical analysis

The results were expressed as the mean±SD. The data were analyzed by ANOVA with Student’s t test and χ2 test. Value of P<0.05 was considered significant.

RESULTS

Characterization of resistance-associated markers in the cells and xenografts

The RT-PCR results demonstrated that mdr1, mrp and lrp mRNAs were detected in the resistant sublines (Fig.1), but the lrp expression level was lower. Flow cytometric analysis was also performed to evaluate the protein level of the MDR-associated genes mdr1, MRP and LRP. The analysis showed that adriamycin induced a significant increase in both P-gp, MRP and LRP expression in the resistant sublines using the MRK16 antibody (P<0.01), the MRPr1 antibody (P<0.01), and the LRP56 antibody (P<0.05, Table 1). The xenografts established with HepG2 adriamycin-resistant sublines can provide a valuable tool to investigate multidrug resistance.

View this table:
Table 1.

Relative expression of resistance-associated markers in HepG2 and HepG2/ADM cells.

Combination of US with adriamycin for the treatment of HepG2/ADM xenografts

When grafted into nude mice, growth of the HepG2/ADM xenografts decreased by 13% as compared to the control (HepG2) group (P>0.05); no prolongation of survival was observed. HepG2/ADM cells, pretreated with adriamycin, expressing Pgp, MRP and LRP, were refractory to the effect of adriamycin and mice bearing this tumor were also resistant to adria-mycin. Adriamycin reduced the growth of HepG2/nude tumors by 73% when the mice were treated with US (P<0.01) and survival of the mice was prolonged 1.6-fold. However growth of the HepG2/nude tumors was slowed only slightly by either US or adriamycin. Similar observations were made with the HepG2/ADM/nude tumors; an anti-tumoral effect of US was obtained by the combination with adriamycin, leading to a growth inhibition of 52% (P<0.01) and a prolonged survival (1.7-fold Fig.2.A, B). These results were all the more striking with HepG2/ADM/nude tumors because they were not inhibited at all by the adriamycin alone (Table 2).

Fig.2.
Fig.2.

Combination of US with adriamycin for the treatment of HepG2/ADM Xenografts. Tumor growth curves of HepG2, a human HepG2 xenografted into nude mice and its variant, HepG2/ADM, obtained from transplanted HepG2/ADM cells. A, control mice with HepG2 were treated with ADM (5 mg/kg) once a week; were treated with US (0.5 W/cm2); or were treated with ADM+US, weekly. B, mice with HepG2/ADM were treated with ADM (5 mg/kg) once a week; were treated with US (0.5 W/cm2); or were treated with ADM+US, weekly.

View this table:
Table 2.

In vivo anti-tumoral effects of US with and without ADM.

mdr1, mrp and lrp mRNA expression

HepG2/ADM xenograft tissue samples were taken from the treatment site in the US-treated animals and from tumors in the sham-treated animals. As shown by RT-PCR, the amplification products of mdr1, mrp, lrp and β-actin were 181 bp, 242 bp, 377 bp and 620 bp, respectively. Over-expression of mdr1 and mrp mRNA was detected in HepG2/ADM/mice, but a low level lrp mRNA expression in HepG2/ADM was also observed. The β-actin signals in HepG2/ADM, HepG2/ADM+ADM, HepG2/ADM+US and HepG2/ADM+ADM+US cells were equally strong, giving a rough estimate that the same amounts of RNA were used in the four compared cell lines. MDR1 mRNA, MRP mRNA and LRP mRNA was determined after exposure to US (0.8 MHz, 0.5 W/cm2, 5 min). The results are shown in Fig. 3. HepG2/ADM (US) showed low expression of MDR1 mRNA and MRP mRNA (P<0.05), the expression of MDR1 mRNA and MRP mRNA decreased approximately 5.5-fold and 3.8-fold, respectively. But LRP mRNA expression of the HepG2/ADM cells showed no significant change (P>0.05), and reached a level close to that of untreated controls.

Fig.3.
Fig.3.

Quantification of mdr1, MRP and LRP mRNA expression by PCR. The ratio between mdr1 or mrp or Irp and β-actin gene is expressed as described in METHODS AND MATERIALS.

Relationship among P-gp, MRP1 and LRP immunostaining

The staining was scored as the percentage of positively stained cells[15]. Results of the immunohistochemistry are summarized in Fig.4. In the control group, 10 of 40 HepG2/ADM xenografts specimens were analyzed, of which 9 (90%) and 7 (70%) showed positive P-gp and MRP expression, respectively. The majority of the samples (80%) were also intermediately positive for LRP, whereas 3 showed only minimal expression. Expression of P-gp was higher than that of LRP (P<0.001). Expression of MRP1 was higher than that of LRP (P<0.001). Expression of P-gp correlated with MRP1 expression: Spearmans’s ρ=0.358 (P<0.01), whereas LRP expression did not correlate with P-gp or MRP1 expression. In contrast, samples that were P-gp and MRP1 negative or sparsely positive were encountered only among the HepG2/ADM+US+ADM samples, but LRP showed no significant change. In the adriamycin-treatment groups, samples had extensive P-gp and MRP immunoreactivity, but none changed significantly in comparion with the HepG2/ADM group (P>0.05). However, there was a significant difference when compared with ultrasound therapy (US) or with the adriamycin plus US group (P<0.05, P<0.001, respectively). Samples that were LRP negative or sparsely positive also comprised all of the HepG2/ADM+ADM groups. There was no significant change in any of the experimental groups (Fig.4.)

Fig.4.
Fig.4.

Relationship among P-gp, MRP1 and LRP immunostaining. a1,2,3,4, immunostaining for P-gp with MRK16 was heterogeneous and mainly localized on the cytoplasmic membrane surface. b1,2,3,4, immunostaining for MRP1 with MRPr1 showed a predominantly granular cytoplasmic staining pattern. c1,2,3,4, immunostaining for LRP with LRP56 was diffuse and cytoplasmic. HepG2/ADM and HepG2/ADM+ADM samples show that both P-gp (a1,2), MRP (b1,2) and LRP (c1,2) are present extensively in the cytoplasmic membranes of the MDR cells, whereas HepG2/ADM+US and HepG2/ADM+ADM+US shows that P-gp (a3,4), MRP (b3,4) are sparsely positive present and LRP (c3,4) is not significantly changed in all the experimental groups, ×400.

DISCUSSION

This study shows that low frequent ultrasound (US) with adriamycin (ADM) administration can be used in the treatment of solid tumors resistant to adriamycin. First, we established the resistant HepG2 xenografts by implanting the HepG2/ADM cells. From Table 1 and Fig.1, it can be seen that both the mRNA and protein level of P-glycoprotein, MRP and LRP increased significantly when HepG2 cells developed resistance. These findings were in accordance with results reported by several authors[16]. Results from our previous study showed that US is able to partially overcome resistance mediated by over-expression of P-glycoprotein in cell culture systems as well as in vivo[17]. Our results demonstrated that low intensity US caused the accumulation of the most and efflux the least adriamycin when compared with HepG2/ADM cells. We also found that low intensity US can have an effect at both the mRNA and protein levels of human hepatoma HepG2/ADM cells.

The use of ultrasound to enhance cancer therapy has been the subject of numerous biological and clinical investigations. In most of these studies, ultrasound has been employed to induce hyperthermia for either direct treatment of small and localized cancerous tumors or as adjuvant therapy to increase the efficacy of radiotherapy and chemotherapy. Lower ultrasound intensities (0.2 to several W/cm2) produce a mild increase in temperature, and enhance the cytotoxicity of radiation therapy and chemotherapy. The precise mechanism for ultrasound-enhanced chemototoxicity is still the subject of debate.

In all tumors tested from various groups in our study, tumor growth inhibition was observed when US treatment in tumor-bearing animals was associated with the ADM, reaching a value of 52% with significant tumor growth delay and prolongation of survival times by 2~3-fold. Such an anti-tumor effect was confirmed in the present study in nude mice bearing HepG2/ADM.

One of the critical elements of medical therapy is effective and targeted delivery of drugs into cells and tissues. The lipid bilayer high-molecular weight molecules into cells[18]. Liu et al.[19] reported that disruption of red blood cell membranes by ultrasound correlates better with the occurrence of stable cavitation. Furthermore, based on the available methodologies to focus ultrasound in the body [20], ultrasound-mediated drug delivery may be targeted to designated regions. Ultrasound-enhanced delivery into cells has been demonstrated in vitro by uptake of extracellular fluid, drugs, and DNA into cells[21] and plant tissues[22].

To date, it is not clear how US signals are transduced to produce a cellular response. This mainly results from the absence of an priori mechanistic hypotheses in studies reporting US biological effects[23]. Despite this, the US effect observed in the current investigation may have resulted from two possible mechanisms. The first relates to US being a form of mechanical stimulation. Given that US energy travels in mechanical waves which carry momentum outward from their source, it may be postulated that the observed mRNA changes occurred as a result of direct cellular mechanical stimulation. This is supported by the observed mRNA response being similar to an mRNA response of MDR cells to mechanical stimuli[24]. Mechanical stimuli may be transduced by the integrin-cytoskeletal system which connects the cell membrane with the nucleus and cytoplasmic constituents of the cell. Alternatively, it may be transduced by the opening of membrane spanning channels. Activation of these processes has the potential to initiate intracellular events stimulating changes in mRNA expression. The second possible mechanism for the observed effect of US in the current investigation is stable cavitation[25] and microstreaming[26]. These responses have been shown to contribute to the in vitro therapeutic effects of US[27].The therapeutic potential of cavitation and microstreaming may be in the generation of shear forces on cellular membranes and alterations in the concentration profiles within the membrane unstirred layers[28]. These effects may lead to altered transport of ions and molecules across the cellular membrane and a subsequent cellular response[29].

Previous studies[30] have demonstrated that ultrasonic cavitation-induced sonochemical reactions were responsible for irreversible inactivation of luciferase and ATPase, and the researchers measured the levels of hydrogen peroxide generated by ultrasound under the conditions of their experiments. In addition to hydrogen peroxide, free radicals generated by ultrasound were responsible for the effects. These effects may account for our results showing that the potent inhibition of P-gp or P190 expression by low intensity US, and intracellular drug concentration was increased and subcellular drug distribution changed, which lead to the reversal of multidrug resistance in the human tumor xenografts.

The current findings suggest that low intensity US had beneficial effects by causing reversal of MDR, by inhibiting tumor growth and prolonging survival time of the tumor-bearing nude mice. US also stimulated expression of the MDR response genes, mdr1 and MRP, and reduced mRNA levels of the multidrug resistant proteins. However, overcoming MDR involves a complex cascade of events. Most of the P-gp modulating agents, currently in use today, are cytotoxic and lack organ specificity due to the abundance of P-gp in other body sites. This application of ultrasound has the potential to replace these agents, and thereby serve as a safer alternative to increase uptake of P-gp substrates in MDR cells as well as to clinically manage MDR. US is well suited for this application because US can be tailored to the specific treatment requirements for a localized concentration of energy deep in tissues by controlling the US of focus, frequency, and duration.

Acknowledgements

We thank the Institute of Ultrasound and Engineering in Medicine, Chongqing University of Medical Science.

Footnotes

  • This work was supported by a grant from the National Natural Science Foundation of China (No. 30200060).

  • Received February 12, 2007.
  • Accepted November 14, 2007.

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