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Year : 2017  |  Volume : 13  |  Issue : 3  |  Page : 533-537

Efficiency of combined blocking of aerobic and glycolytic metabolism pathways in treatment of N1-S1 hepatocellular carcinoma in a rat model

1 Department of Radiology, Division of Interventional Radiology and Image Guided Therapy, Memorial Sloan Kettering Cancer Center, New York 10065, USA
2 Department of Radiology, University of Virginia, Charlottesville, Virginia, USA
3 Department of Radiology, Case Western Reserve University, Cleveland, Ohio 44106-5056, USA
4 Department of Radiology, Case Western Reserve University, Case Center for Imaging Research, Cleveland, Ohio 44106-5056, USA

Date of Web Publication31-Aug-2017

Correspondence Address:
Hooman Yarmohammadi
Department of Radiology, Division of Interventional Radiology and Image Guided Therapy, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York 10065
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0973-1482.172127

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 > Abstract 

Background/Aim: The aim of this study was to determine whether the addition of bumetanide (BU), a glycolytic metabolism pathway inhibitor, to arterial embolization improves tumor necrosis of N1-S1 hepatocellular carcinoma in a rat model.
Materials and Methods: N1-S1 tumors were surgically implanted in the liver of 14 Sprague-Dawley rats. The rats were divided into three groups: In control group (n = 5), 1 ml of normal saline was injected intra-arterially. The tumor in the transarterial embolization group (TAE, n = 4) was embolized using 10 mg of 50–150 μ polyvinyl alcohol (PVA) particles and embolization plus BU group (TAE + BU, n = 5) were embolized with 10 mg of PVA plus 0.04 mg/kg of BU. Tumor volume was measured using two-dimensional ultrasound before intervention and twice a week afterward. Relative tumor volume after the intervention was calculated as the percentage of preinterventional tumor volume. After 4 weeks of observation, the rats were sacrificed for histopathological evaluation.
Results: No statistically significant difference was detected in the preintervention tumor sizes between the three groups (P > 0.05). In the control group, the relative tumor volume increased to 142.5% larger than baseline measurements. In the TAE group, the tumor volume decreased by 18.2 ± 12.2%. The tumor volume in the TAE + BU group decrease by 90.4 ± 10.2%, which was 72.2% more than in TAE only group (P < 0.0001). Histopathological evaluation demonstrated no residual tumor in the TAE + BU group.
Conclusion: Tumor necrosis significantly increased in N1-S1 tumor that received BU at the time of TAE when compared to TAE alone.

Keywords: Aerobic metabolism, bumetanide, glycolytic inhibition, hepatocellular carcinoma, N1-S1, transarterial embolization

How to cite this article:
Yarmohammadi H, Wilkins LR, Erinjeri JP, Novak RD, Exner AA, Wu H, Petre EN, Boas E, Ziv E, Haaga JR. Efficiency of combined blocking of aerobic and glycolytic metabolism pathways in treatment of N1-S1 hepatocellular carcinoma in a rat model. J Can Res Ther 2017;13:533-7

How to cite this URL:
Yarmohammadi H, Wilkins LR, Erinjeri JP, Novak RD, Exner AA, Wu H, Petre EN, Boas E, Ziv E, Haaga JR. Efficiency of combined blocking of aerobic and glycolytic metabolism pathways in treatment of N1-S1 hepatocellular carcinoma in a rat model. J Can Res Ther [serial online] 2017 [cited 2020 Sep 25];13:533-7. Available from: http://www.cancerjournal.net/text.asp?2017/13/3/533/172127

 > Introduction Top

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide.[1] Up to 75% of patients are not surgical candidates based either on extend of the disease or underlying cirrhosis. Transarterial embolization (TAE) and chemoembolization (TACE) have been used extensively in patients with intermediate-stage disease (Barcelona Clinic Liver Cancer Class B or Okuda Stage I or II) and have been able to increase overall survival when compared to best supportive care.[2],[3],[4] A major tumoricidal mechanism in both TAE and TACE is the ischemic effect caused by occlusion of the arterial blood supply.[5],[6] The goal during TAE is to achieve intra-tumoral small vessel occlusion that will result in severe hypoxia or anoxia.[7] Severe hypoxia or anoxia will block the aerobic pathway of tumor metabolism and causes cell death via a hypoxia-inducible factor 1-independent pathway.[8]

Despite high success rate observed in TAE and TACE, there is a high rate of local recurrence that may be from residual viable tumor which survives the ischemic event. This is likely because we are only targeting the aerobic pathways of tumor cells while the survival of residual tumor cells occurs through the anaerobic/glycolytic metabolism pathways. Tumor cells microenvironment differs from normal cell and they mainly rely on the anaerobic pathway of oxygen-independent glycolysis, long known as the “Warburg effect.”[9] Therefore when treating HCC with TAE, only tumor cells that are sensitive to hypoxia are targeted and those that are resistant to hypoxia and predominantly rely on oxygen-independent glycolysis will survive.

Bumetanide (BU) (3-n-butylamino-4-phenoxy-5-sulfamylbenzoic acid; Validus Pharmaceuticals LLC, Parsippany, NJ, USA) is a loop diuretic with a rapid onset and short duration of action.[10],[11] This drug is Food and Drug Administration approved to treat edema and is most commonly used in patients with congestive heart failure.[10],[11] BU interferes with renal cAMP and/or inhibits the sodium-potassium ATPase pump.[11] In addition, BU blocks transport of chloride across the cancer cells that will ultimately lead to blockage of transport of HCO3 into the cells.[12],[13] Therefore, the intra-cellular pH decreases making the internal milieu acidic which blocks the rate limiting step of phosphofructose kinase. Blocking the rate limiting step of anaerobic glycolysis will ultimately result in tumor cell death.

The aim of this study was to evaluate if addition of a glycolytic inhibitor (BU) to TAE will increase tumor necrosis of N1-S1 HCC in a rat model.

 > Materials and Methods Top

This study was approved by the Institutional Animal Care and Use Committee at our institution. In addition, all procedures were in compliance with the National Institute of Health's Guide for the Care and Use of Laboratory Animals. Animals were anesthetized using 2–5% isoflurane by mask. The animals were monitored throughout the procedures and were allowed to recover after each procedure. Analgesia after surgery was administered with buprenorphine (0.01–0.05 mg/kg subcutaneously every 8–12 h) as needed for pain. Euthanasia was performed by over-breathing carbon dioxide.

N1-S1 cells (ATCC, Manassas, Virginia, USA) were maintained in suspension culture flasks at 37°C and 5% CO2 until needed for inoculation. To maintain the culture, cells were allowed to proliferate in Iscove's modified Dulbecco's Medium (Catalog No. 30-2005, ATCC) with 5% fetal bovine serum and passed biweekly. To establish liver tumors, 5 × 106 viable cells suspended in 0.2 ml incomplete medium were inoculated under the capsule of the medial aspect of the left lobe of the liver of Sprague-Dawley rats (6 weeks old, Charles River, average weight, 200–250 g) after a mini-laparotomy. Tumors were allowed to grow for 14 days.

After 14 days, tumors were evaluated using a two-dimensional ultrasound (US). The tumor size was measured in three dimensions of length (L), width (W), and height (H). Measurements were performed right before the interventions and twice a week for 4 weeks. Tumor volume (V) was calculated by the formula: V = 0.5 × L × W × H. Relative volume after the intervention was calculated as the percentage of preintervention tumor volume.

After anesthesia had been established, the upper abdomen was shaved and cleaned with iodine solution. Sterile towels were applied, and intravenous access was established. A 2 cm midline laparotomy was performed. The gastroduodenal artery (GDA) was dissected using a stereomicroscope and cannulated in a retrograde fashion with PE-50 polyethylene tubing. The common hepatic artery was temporarily occluded using a clip.

Tumors were divided into three groups: (1) In the control group (n = 5), 1 ml normal saline (NS) was injected; (2) in the arterial embolization group (TAE, n = 4), 10 mg of 50–150 um polyvinyl alcohol (PVA) particles in 1 ml NS was injected; (3) in the third group (TAE + BU, n = 5), the tumor was embolized using 10 mg of 50–150 um PVA particles plus 0.04 mg/kg BU in 1 ml NS. All of these doses were injected slowly and over 1 min. The doses were all determined utilizing preliminary data. After the intervention, the GDA was ligated and abdominal wall incision was closed. The animals were allowed to recover.

In Groups 2 and 3, a contrast-enhanced US (CE-US) was performed before intervention to establish blood flow to tumor. After embolization, another CE-US was performed to confirm complete cessation of tumor blood flow using a previously established technique.[14]

After 4 weeks, the animals were euthanized and both normal liver and the tumors were evaluated for histopathological changes. Hematoxylin and eosin was used to stain the tissues. Tumor viability was estimated by light microscopic examination to quantitate the antitumoral effects of each intervention. Histopathological evaluation was performed by an independent pathologist who was blinded to the interventions performed.

Statistical analysis

Tumor sizes (comparison to 100% at T0 =100) were analyzed and compared between three groups. P < 0.05 were considered statistically significant. The repeated measures analysis of variance was used to simultaneously compare the difference in tumor volume between treatment groups and over time. Potential specificity problems were encountered by employing the Greenhouse–Geisser correction and when necessary the logarithmic function was used to transform variable data to establish normality prior to analysis.

 > Results Top

There was no significant difference in the initial tumor sizes between each group (P > 0.05). Interventions and/or embolization were successfully performed in all three groups. CE-US demonstrated successful stasis and secession of blood flow to the tumors after embolization in TAE and TAE + BU group.

In the control group, tumors showed a relative tumor volume that was 142.5% larger when compared with the baseline measurements [Figure 1]. In the TAE group, the tumor volume decreased by 18.2 ± 12.2% [Figure 1]. Tumor volume demonstrates mild initial increase but decreases afterward. This is most likely related to initial post embolization tumor cell edema and inflammation which rapidly subsides. The tumor volume in the TAE + BU group decrease by 90.4 ± 10.2% which was 72.2% more decrease compared to the TAE group (P < 0.001) [Figure 1].
Figure 1: Tumor relative volume by weeks post procedure. Transarterial embolization + bumetanide group demonstrates a statistically significant decrease in tumor size when compared to the control group and the transarterial embolization only group (P < 0.0001)

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Histopatholgic analysis demonstrated tumor growth and viable tumor cells in the control group as expected [Figure 2]. In this group, tumor has geographic necrosis, and there is proliferation of peritumoral bile ductules. Background liver shows portal triads with mild inflammation and extensive bile ductule proliferation. In the second group that tumors were embolized by TAE only, small amount of residual viable tumors was seen in 2 out of 5 rats [Figure 3]. In the TAE + BU group, no residual viable tumor was seen, and areas of fibrosis, necrosis, and dystrophic calcification were observed. In the background liver, there was no inflammation or bile duct proliferation and no mononucleated giant cell reaction [Figure 4].
Figure 2: The histopathologic slides of a specimen collected from the control group in which the tumor was treated with only normal saline. This image demonstrates viable tumor (black star) with areas of necrosis (black arrow). The background liver shows portal triad with mild inflammation and extensive bile ductule proliferation (black arrow head)

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Figure 3: The histopathologic slides of a specimen from the transarterial embolization group in which the tumor was treated with embolization only using polyvinyl alcohol. This image demonstrates mainly areas of tumor necrosis (black star) with some viable tumor cells (black arrow)

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Figure 4: The histopathologic slides of a specimen from the transarterial embolization + bumetanide group in which the tumor was treated with embolization plus bumetanide. There are areas of fibrosis, necrosis, and dystrophic calcification (black arrow head) where the tumor existed (black star). There is no residual tumor. Background liver demonstrates no evidence of inflammation or bile duct proliferation. Giant cells are seen in the periphery of the tumor (black arrow)

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 > Discussion Top

Compared to normal cells, cancer cells have greater acidic burden which requires buffering with HCO3. Normal cells use carbonic anhydrase to provide the HCO3, and this process occurs in the cytoplasm. Aggressive cancer cells use two different carbonic anhydrases, CAIX and CAXII, which reside on the outer surface of the cell membrane. When CO2 and water are converted to H+ and HCO3, it occurs outside the cell. The HCO3 is exchanged via the CL/HCO3 mechanism. BU blocks transport of Cl into the cell and consequently HCO3 cannot enter the cancer cells. This will results in decrease in the intra-cellular pH making the internal milieu acidic which blocks the rate limiting step of phosphofructose kinase. Once this step is blocked, the cancer cell glycolytic pathway is inhibited.

The current study demonstrates that addition of BU to TAE was associated with 72.2% increase in tumor shrinkage/necrosis when compared to TAE alone and 90.4% when compared to baseline volume prior to treatment. This effect is because BU blocks tumor cell glycolytic pathways. Therefore while TAE causes hypoxic/ischemic effect and blocks oxidative phosphorylation (aerobic metabolism pathways), BU blocks glycolysis causing death in those tumor cells that are resistant to hypoxia and are more dependent on glycolytic pathways, hence better response to embolization. This combined approach is especially helpful when considering that tumor cells utilize both oxidative phosphorylation and glycolysis.

The present study proves that the combining hypoxia with glycolytic inhibitors will increase the antitumor effect of ischemia. Targeting two aspects of metabolism has been investigated and Gang et al. have used this method in treating breast cancer.[15]

Inhibition of the glycolytic pathway in cancer cell as a treatment option has been previously studied with other drugs.[16],[17],[18] One of these drugs is 3-bromopyrovate (3-BrPA). This drug inhibits hexoskinase 2 through cessation of adenosine triphosphate production. Hexoskinase 2 is the first step of glycolysis; therefore by blocking this enzyme, both oxidative phosphorylation and glycolysis will be blocked.[16],[17],[18] Early reports on intra-arterial injection of 3-BrPA demonstrated a dose-dependent response with significant success in decreasing viable tumor.[16] However, Shin et al., in a most recent study on VX2 rabbit model, was not able to show significant difference between injecting NS and 3-BrPA.[17] In addition, Chang et al. demonstrated the 3-BrPA is associated with significant toxicity to the surrounding normal liver parenchyma and the gastrointestinal system.[18] In the present study, all rabbits in the TAE + BU group survived the 4 weeks of observation. In addition, the histopathological examination did not show any signs of toxicity in the surrounding liver parenchyma. As seen in [Figure 4], there was no inflammation or bile duct proliferation and no mononucleated giant cell reaction in the background liver in this group. This is in contrast with the histopathological findings in TAE group which demonstrated inflammation in the surrounding liver tissue [Figure 3]. Compared to Chang et al. study, in which 12 of 20 rabbits died within 3 days of receiving intra-arterial 3-BrPA, BU appears to be a safer treatment option.[18]

Another group of glycolytic inhibitors block the lactate dehydrogenase (LDH) that is the last glycolytic enzyme that catalyzes the reduction of pyruvate to lactate. Oxamate is a LDH-inhibitor of both A and B subunits of LDH.[19]

Lack of peritumoral inflammation in the normal liver tissue is a significant finding. While the exact reason is not certain, it may be hypothesis that the blockage of glycolysis pathways may have had a major role.

One of the limitations of this study is that the intra-cellular pH was not measured to document the proposed mechanism of action of BU, which is causing significant intra-cellular acidosis in cancer cells. The second limitation of this study was the small number of animals enrolled in the control and interventional group. The strong point of the current study is that N1-S1 tumor is a true rat hepatocellular tumor. However while the N1-S1 tumor is a true rat HCC, it was implanted in a normal liver. This is in contrary to what is observed in clinical practice, in which HCC is most commonly seen in a cirrhotic liver.

 > Conclusion Top

The addition of BU to tumor embolization is able to significantly improve tumor response when compared to embolization alone in a rat N1-S1 tumor model.


The authors would like to thank the National Institutes of Health, the Cancer Center Program for funding this study.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

 > References Top

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Llovet JM, Brú C, Bruix J. Prognosis of hepatocellular carcinoma: The BCLC staging classification. Semin Liver Dis 1999;19:329-38.  Back to cited text no. 4
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Olesen KH, Sigurd B, Steiness E, Leth A. Bumetanide, a new potent diuretic. A clinical evaluation in congestive heart failure. Acta Med Scand 1973;193:119-31.  Back to cited text no. 10
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Kahle KT, Staley KJ. The bumetanide-sensitive Na-K-2Cl cotransporter NKCC1 as a potential target of a novel mechanism-based treatment strategy for neonatal seizures. Neurosurg Focus 2008;25:E22.  Back to cited text no. 12
Ju M, Scott-Ward TS, Liu J, Khuituan P, Li H, Cai Z, et al. Loop diuretics are open-channel blockers of the cystic fibrosis transmembrane conductance regulator with distinct kinetics. Br J Pharmacol 2014;171:265-78.  Back to cited text no. 13
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Geschwind JF, Ko YH, Torbenson MS, Magee C, Pedersen PL. Novel therapy for liver cancer: Direct intraarterial injection of a potent inhibitor of ATP production. Cancer Res 2002;62:3909-13.  Back to cited text no. 16
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Chang JM, Chung JW, Jae HJ, Eh H, Son KR, Lee KC, et al. Local toxicity of hepatic arterial infusion of hexokinase II inhibitor, 3-bromopyruvate:In vivo investigation in normal rabbit model. Acad Radiol 2007;14:85-92.  Back to cited text no. 18
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]


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