|Year : 2018 | Volume
| Issue : 1 | Page : 3-7
A prospective study on the effects of therapeutic ultrasound in cancer using an animal model
Chinthalapalli Siva Ram1, Durg Vijay Rai2, M Jayanand3, Rajendra Kumar Saxena4, Maya Dutt Joshi5, Sonali Gangwar5
1 Department of Physiotherapy, I. T. S Paramedical College, Muradnagar, Ghaziabad, Uttar Pradesh, India
2 Centre for Biological Engineering, Shobhit University, Saharanpur, Uttar Pradesh, India
3 Centre for Research and Innovation, Noida International University, Noida, Uttar Pradesh, India
4 Center for Biomedical Engineering, Indian Institute of Technology, New Delhi, India
5 Centre for Biomedical Engineering, Shobhit University, Meerut, Uttar Pradesh, India
|Date of Submission||11-Dec-2017|
|Date of Acceptance||19-Dec-2017|
|Date of Web Publication||19-Jun-2018|
Dr. Chinthalapalli Siva Ram
I.T.S Institute of Health and Allied Sciences, Delhi-Meerut Road, Muradnagar, Ghaziabad - 201 206, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
CONTEXT: Ultrasound is emerging as a novel treatment agent for cancer. The advantage of using ultrasound is that it is not an electromagnetic radiation; hence, it does not produce the undesired harmful effects encountered through the repeated use of electromagnetic radiation.
AIMS: The present study was aimed to evaluate the therapeutic potential of ultrasound in 7,12-dimethyl benz (a) anthracene (DMBA)-induced sarcoma in rats.
SETTINGS AND DESIGN: Forty female Wistar rats were used in the experimental study. They were allocated into four groups. DMBA was used to induce sarcoma in 20 rats. Therapeutic ultrasound was applied at 2.5 W/cm2 for 10 min (continuous mode) to 10 sarcoma tumor-bearing rats and normal 10 rats.
SUBJECTS AND METHODS: DMBA was used to induce sarcoma in rats. Body weight, tumor weight, and serum enzymes were determined following treatment with therapeutic ultrasound (Chattanooga Group, Hixson,TN USA (Model: Intelect® Mobile Combo Model No. 2778).
STATISTICAL ANALYSIS USED: Statistical analysis was performed using SPSS (SPSS Inc., Chicago, IL, USA) statistical package. The results were expressed as mean, standard error of mean (SEM). The one-way analysis of variance followed by post hoc test least significant difference was used to correlate the difference between the variables. Values were considered statistically significant if P < 0.05.
RESULTS: There were significant increases on the body weight and tumor weight of treated rats. The increased activities of serum pathophysiological enzymes aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, ACP, and lactate dehydrogenase of ultrasound-treated rats were significantly (P < 0.05) higher than the control levels indicating loss of redox homeostasis. The histopathological analysis of sarcoma tissues showed extensive hemorrhage and necrosis indicating the antitumor nature of ultrasound.
CONCLUSIONS: The results of the present study indicate that ultrasound significantly suppresses DMBA-induced sarcoma in rats.
Keywords: Cancer, sarcoma, therapeutic, ultrasound
|How to cite this article:|
Ram CS, Rai DV, Jayanand M, Saxena RK, Joshi MD, Gangwar S. A prospective study on the effects of therapeutic ultrasound in cancer using an animal model. Physiother - J Indian Assoc Physiother 2018;12:3-7
|How to cite this URL:|
Ram CS, Rai DV, Jayanand M, Saxena RK, Joshi MD, Gangwar S. A prospective study on the effects of therapeutic ultrasound in cancer using an animal model. Physiother - J Indian Assoc Physiother [serial online] 2018 [cited 2019 Jan 15];12:3-7. Available from: http://www.pjiap.org/text.asp?2018/12/1/3/229115
| Introduction|| |
Ultrasound cavitation leads to the formation of reactive oxygen species (ROS) and its consequences are of primary interest. Recent clinical studies have demonstrated that cancer cells can be targeted and destroyed by a single blast of ultrasound. However, the extent to which ultrasound affects cancerous tissue is an area of ongoing research and needs to be explored further. The biophysical effects of therapeutic ultrasound have been examined through in vitro studies. Extrapolation of these results to humans is, therefore, conjectural. Our study aimed to study the effect of ultrasound therapy on morphological, biochemical, and histopathological changes in sarcoma cancer in rats.
| Subjects and Methods|| |
Virgin female Wistar rats, 7 weeks of age, were purchased from Central Animal House, AIIMS, New Delhi, and were used in the experiment. The experimental design was performed in accordance with the current ethical norms approved by the CPCEA Government of India and Institutional Animal Ethics Committee Guidelines and approved by the Institutional Ethics Committee vide approval No. IAEC No: ITS/01/IAEC/2013. 7,12-dimethyl benz (a) anthracene (DMBA) is a known carcinogen which produces mammary cancer and sarcomas in rats. DMBA was purchased from Sigma chemical company (St. Louis, MO, USA). All other chemicals used were of analytical grade procured from local commercial sources. The carcinogen mixture was prepared in biosafety Level II laboratory conditions. The rats were divided into four groups of ten rats each as follows: Group 1: normal control rats, Group 2: control rats administered with ultrasound therapy (2.5 W/cm 2), Group 3: DMBA-induced sarcoma cancer rats with sham treatment, and Group 4: DMBA-induced sarcoma cancer rats administered with ultrasound therapy 2.5 W/cm 2, continuous output). A single dose of DMBA (25 mg/kg bw/rat) in 0.5 ml of corn oil was injected into the rat abdomen. After the tumor had grown (over 3–4 weeks) to a minimum size of 1 cm in at least one dimension, the tumor was insonated with a physiotherapy ultrasound machine (Chattanooga Corp, USA) at 1 MHz, 2.5 W/cm 2, continuous output.
Blood was collected, and the serum was centrifuged at 5000 rpm for 15 min to obtain a clear supernatant for use in further biochemical analysis. The total body weight gain of the control and experimental animals was recorded periodically throughout the experimental period. Enzymes levels of oxidants and antioxidants in serum were determined by assay. The tumor tissue was immediately fixed in 10% neutral buffered formalin, embedded in paraffin, 5 μm section was cut using a microtome, and then, rehydrated with xylene and graded series of ethanol. The specimens were then stained with hematoxylin and eosin. The H and E-stained specimens were examined by a pathologist to histopathologically classify the tumors as described by Royal College of Pathologists UK.
| Results|| |
Analysis of body weight and tumor weight of rats
[Table 1] and [Graph 1] show the body weight of control and experimental rats. The body weight of control Group I rats (219.46 g) was significantly higher as compared to Group III rats (154.33 g) following DMBA treatment (P< 0.05). The body weight of DMBA-induced Group IV rats following ultrasound therapy treatment was significantly higher (176.21 g) as compared to Group III rats (P< 0.05). However, no statistically significant changes could be observed in the body weight of Group II rats treated with ultrasound therapy (211.82 g) as compared to control Group I rats (219.46 g) (P > 0.05).
|Table 1: Effect of ultrasound therapy treatment on total body weight and tumor weight of control and 7,12-dimethyl benz(a)anthracene-treated rats|
Click here to view
Effect of ultrasound therapy on serum pathophysiological enzymes
[Table 2] and [Graph 2] show the serum level of pathophysiological enzymes aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) in control and experimental rats. The serum level of pathophysiological enzymes in control Group II rats following ultrasound therapy treatment was significantly increased as compared to control Group I rats (P< 0.05). Ultrasound therapy treatment to DMBA-induced Group IV rats following ultrasound therapy treatment significantly increased the level of pathophysiological enzymes as compared to Group III rats (P< 0.05). The treatment of DMBA-induced rats with ultrasound therapy significantly increased the level of serum pathophysiological enzymes, namely, AST from 395.41 to 686.87 U/L, ALT from 95.65 to 422.22 U/L, ALP from 285.64 to 1242 U/L, and LDH from 179.90 to 501.48 U/L (P< 0.05).
|Table 2: Effect of ultrasound therapy on serum pathophysiological enzymes|
Click here to view
Effect of ultrasound therapy on serum antioxidants
The levels of serum antioxidants, namely, total glutathione (GSH), Vitamin C, and Vitamin E are presented.
[Table 3] and [Graph 3] show the level of nonenzymatic antioxidants GSH, Vitamin C, and Vitamin E in control and experimental rats in serum. The serum level of antioxidants in control Group II rats following ultrasound therapy treatment was significantly lower as compared to control Group I rats (P< 0.05). Ultrasound therapy treatment to DMBA-induced Group IV rats significantly lowered the level of antioxidants as compared to Group III rats (P< 0.05). The treatment of DMBA-induced rats with ultrasound therapy significantly lowered the level of serum antioxidants, namely, GSH from 7.42 U/L to 0.43 U/L, Vitamin C from 3.54 U/L to 0.08 U/L, and Vitamin E from 1.2 U/L to 0.05 U/L (P< 0.05).
Histopathological analysis of tumor tissue
[Figure 1]a,[Figure 1]b,[Figure 1]c,[Figure 1]d shows histological images of sarcoma tissue in control and experimental group of rats.
|Figure 1: (a) Control rats showed normal architecture, i.e. hyalinized stroma composed of round cell population. (b) Control + ultrasound therapy-treated animals exhibited marked area of necrosis (outlined by light green line) surrounded by mixed inflammatory infilterate (outlined by black line) of acute and chronic cells. (c) 7,12-Dimethyl benz (a) anthracene-induced tumor rats showed sarcoma tumor with loss of normal architecture. Round cell population showing marked anaplastic features of pleomorphism, hyperchromatism, anisonucleosis, and anisocytoses with 4–5 mitotic figures. Prominent vasculature (marked by yellow arrows) can be appreciated with proliferating endothelial cells. (d) 7,12-Dimethyl benz (a) anthracene and ultrasound therapy-treated rats in addition to all the features of (c) above showed marked areas of necrosis (outlined by black line and arrow) and haemorrhage|
Click here to view
| Discussion|| |
Various mechanisms contribute to weight loss of the host in cancerous condition. No significant changes could be observed in the final body weight of control Group II rats treated with ultrasound therapy in comparison to control Group I rats. This shows that ultrasound therapy in control group did not alter the anabolic metabolism of the rats. Group 3 (DMBA) rats and in Group 4 (DMBA + ultrasound) rats showed a significant reduction in body weight. This is because cancer causes cachexia, i.e. generalized weight loss. On comparison, Group 4 rats showed positive effect of ultrasound therapy by a significant reduction in tumor volume. We believe that it may be due to the arrest of tumor progression in Group 4 rats.,
In our study, there was a marked increase in serum concentrations of pathophysiological enzymes in Group 2, Group 3, and Group 4 in comparison to Group 1. These changes further exacerbate the whole body inflammatory response into vicious cycle of accelerating organ dysfunction. We postulate that the increase in serum and liver prooxidant enzymes is attributable to the thermal effects and chemical effects caused by ultrasound on normal cells.
Ultrasound therapy can cause rapid increase in ROS levels even in normal Group 2 cells. This increase may be due to an increase in lipid peroxidation and resultant increase in plasma levels of lipid peroxide after a thermal injury.,,
The elevated levels of prooxidant enzymes in Group 3 (DMBA group) indicate that there was an oxidative stress environment within the cancerous cells. This is in agreement with studies which show that a moderate increase in ROS can promote cell proliferation and differentiation.,
DMBA is a chemical carcinogen and causes gradual changes in the redox homeostasis of the cells whereas ultrasound therapy causes sudden oxidative stress. The oxidative stress response in Group 2 caused damage to cells in the form of necrosis. The rapid increase in oxidative stress within a short span of time may lead to cellular damage as postulated by many studies.
Ultrasound therapy to cancer cells caused an exorbitant rise in the level of oxidative enzymes and leads to cell death. We believe that this is because redox homeostasis within the cancerous cells has been lost, and hence, the cancerous cells may not be able to produce antioxidants at a rate required to neutralize the oxidative stress during exposure to ultrasound therapy. In Group 2 (control + ultrasound group), the level of oxidative stress enzymes was lower because the normal cells can produce some amounts of antioxidant enzymes which can neutralize the oxidative stress enzymes [Table 2] and [Figure 2].,
There was cellular damage, hemorrhage, and necrosis after ultrasound therapy. These effects range from hemorrhage to complete cellular disruption. This is attributable to the collapse of bubbles of inertial cavitations. Sonication can trigger apoptosis in both normal and malignant cells. Our findings revealed that low-intensity ultrasound markedly kills cells by damaging the ultrastructure and morphology.
In our study, the sarcoma tumor was particularly susceptible to ultrasonic therapy. This correlated well with other in vitro studies., Emerging evidence has confirmed that low-intensity ultrasound markedly inhibits the proliferation and clone formation of tumor cells through heat, mechanical effects, and acoustic cavitation. Cellular necrosis may be due to autophagy in the tumors. The induction of apoptosis by ultrasound therapy may lead to a substantial improvement in antitumor therapy.
| Conclusions|| |
The results of the present study clearly establish the anticancer efficacy of ultrasound therapy against DMBA-induced sarcoma in rats. In addition, the alteration in the levels of tumor biomarker marker enzymes indicates the antitumor activity of ultrasound therapy. Our results underlie the potency of ultrasound therapy as an effective therapeutic agent in the treatment of cancer. However, further studies are warranted to elucidate the exact molecular mechanism underlying the action of ultrasound in reducing the toxic effects of DMBA in sarcoma cancer.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Baker KG, Robertson VJ, Duck FA. A review of therapeutic ultrasound: Biophysical effects. Phys Ther 2001;81:1351-8.
Prentice P, Cuschieri A, Dholakia K, Prausnitz M, Campbell P. Membrane disruption by optically controlled cavitation. Nat Phys 2005;1:107-10.
Husseini GA, Pitt WG. The use of ultrasound and micelles in cancer treatment. J Nanosci Nanotechnol 2008;8:2205-15.
O'Brien WD. Thermal and non-cavitational mechanisms. In: Frenkel V. Therapeutic Ultrasound: Mechanism to Applications. Ch. 2. New York: Nova Science Publishers; 2011. p. 7-38.
Flesher JW, Sydnor KL. Carcinogenicity of derivatives of 7,12-dimethylbenz(a)anthracene. Cancer Res 1971;31:1951-4.
Stein TP. Cachexia, gluconeogenesis and progressive weight loss in cancer patients. J Theor Biol 1978;73:51-9.
Keshavarzi A, Vaezy S, Noble ML, Chi EY, Walker C, Martin RW, et al.
Treatment of uterine leiomyosarcoma in a xenograft nude mouse model using high-intensity focused ultrasound: A potential treatment modality for recurrent pelvic disease. Gynecol Oncol 2002;86:344-50.
Huber PE, Debus J. Tumor cytotoxicity in vivo
and radical formation in vitro
depend on the shock wave-induced cavitation dose. Radiat Res 2001;156:301-9.
Gibran NS, Heimbach DM. Current status of burn wound pathophysiology. Clin Plast Surg 2000;27:11-22.
Riesz P, Kondo T. Free radical formation induced by ultrasound and its biological implications. Free Radic Biol Med 1992;13:247-70.
Cetinkale O, Belce A, Konukoglu D, Senyuva C, Gumustas MK, Tas T, et al.
Evaluation of lipid peroxidation and total antioxidant status in plasma of rats following thermal injury. Burns 1997;23:114-6.
Sun X, Xu H, Shen J, Guo S, Shi S, Dan J, et al.
Real-time detection of intracellular reactive oxygen species and mitochondrial membrane potential in THP-1 macrophages during ultrasonic irradiation for optimal sonodynamic therapy. Ultrason Sonochem 2015;22:7-14.
Yumita N, Iwase Y, Watanabe T, Nishi K, Kuwahara H, Shigeyama M, et al.
Involvement of reactive oxygen species in the enhancement of membrane lipid peroxidation by sonodynamic therapy with functionalized fullerenes. Anticancer Res 2014;34:6481-7.
Barrera G. Oxidative stress and lipid peroxidation products in cancer progression and therapy. ISRN Oncol 2012;2012:137289.
Toyokuni S, Okamoto K, Yodoi J, Hiai H. Persistent oxidative stress in cancer. FEBS Lett 1995;358:1-3.
Filomeni G, De Zio D, Cecconi F. Oxidative stress and autophagy: The clash between damage and metabolic needs. Cell Death Differ 2015;22:377-88.
Wang X, Liu Q, Wang Z, Wang P, Hao Q, Li C, et al.
Bioeffects of low-energy continuous ultrasound on isolated sarcoma 180 cells. Chemotherapy 2009;55:253-61.
Nathan FM, Singh VA, Dhanoa A, Palanisamy UD. Oxidative stress and antioxidant status in primary bone and soft tissue sarcoma. BMC Cancer 2011;11:382.
Prieur F, Pialoux V, Mestas JL, Mury P, Skinner S, Lafon C, et al.
Evaluation of inertial cavitation activity in tissue through measurement of oxidative stress. Ultrason Sonochem 2015;26:193-9.
Feng Y, Tian Z, Wan M. Bioeffects of low-intensity ultrasound in vitro
: Apoptosis, protein profile alteration, and potential molecular mechanism. J Ultrasound Med 2010;29:963-74.
Lagneaux L, de Meulenaer EC, Delforge A, Dejeneffe M, Massy M, Moerman C, et al.
Ultrasonic low-energy treatment: A novel approach to induce apoptosis in human leukemic cells. Exp Hematol 2002;30:1293-301.
Wang P, Leung AW, Xu C. Low-intensity ultrasound-induced cellular destruction and autophagy of nasopharyngeal carcinoma cells. Exp Ther Med 2011;2:849-52.
Ivone M, Pappalettere C, Watanabe A, Tachibana K. Study of cellular response induced by low intensity ultrasound frequency sweep pattern on myelomonocytic lymphoma U937 cells. J Ultrasound 2016;19:167-74.
Wang X, Liu Q, Wang P, Wang Z, Tong W, Zhu B, et al.
Comparisons among sensitivities of different tumor cells to focused ultrasound in vitro
. Ultrasonics 2009;49:558-64.
Feril LB Jr., Kondo T, Cui ZG, Tabuchi Y, Zhao QL, Ando H, et al
. Apoptosis induced by the sonomechanical effects of low intensity pulsed ultrasound in a human leukemia cell line. Cancer Lett 2005;221:145-52.
Sasnauskiene A, Kadziauskas J, Vezelyte N, Jonusiene V, Kirveliene V. Apoptosis, autophagy and cell cycle arrest following photodamage to mitochondrial interior. Apoptosis 2009;14:276-86.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]