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 Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 11  |  Issue : 4  |  Page : 823-829

Doxorubicin enhances 131 I-rituximab induced cell death in Raji cells


1 Isotope Applications and Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai, Maharashtra, India
2 Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai, Maharashtra, India
3 Department of Nuclear Sciences and Nuclear Applications, IAEA, Vienna, Austria

Date of Web Publication15-Feb-2016

Correspondence Address:
G Samuel
Isotope Applications and Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai - 400 085, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.140844

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 > Abstract 

Aim: There are various therapeutic modalities of treatment for non-Hodgkin's lymphoma, but with certain limitations, hence, investigating the scope of combined therapeutic approach.
Materials and Methods: In this article, cellular toxicity, apoptosis and expression of mitogen-activated protein kinase signaling pathway proteins were investigated in Raji cells preincubated with doxorubicin followed by 131 I-rituximab (rituximab radiolabeled with Iodine-131) treatment.
Results: It was found that the 131 I-rituximab in combination with doxorubicin showed a higher amount of cell toxicity and apoptosis compared to respective controls. Expression of anti-apoptotic protein (B-cell lymphoma-extra-large) was downregulated and cleavage of poly (ADP-ribose) polymerase, a marker of apoptosis was higher in cells treated with doxorubicin (2 μg/mL) and 131 I-rituximab (P ≤ 0.05). Moreover, in these cells the basal level of expression of p42/44 and p38 were increased while its phosphorylation was decreased.
Conclusion: These results suggest that doxorubicin has the potential to sensitize 131 I-rituximab induced cell death in Raji cells.

Keywords: Apoptosis, doxorubicin, 131 I-rituximab, mitogen-activated protein kinase, rituximab


How to cite this article:
Kumar C, Pandey B N, Samuel G, Venkatesh M. Doxorubicin enhances 131 I-rituximab induced cell death in Raji cells. J Can Res Ther 2015;11:823-9

How to cite this URL:
Kumar C, Pandey B N, Samuel G, Venkatesh M. Doxorubicin enhances 131 I-rituximab induced cell death in Raji cells. J Can Res Ther [serial online] 2015 [cited 2019 Sep 20];11:823-9. Available from: http://www.cancerjournal.net/text.asp?2015/11/4/823/140844


 > Introduction Top


Non-Hodgkin's lymphoma (NHL), a group of hematological malignancies of which >90% originate from the B-cells, [1] is the eleventh most common cause of cancer incidence [2] and ranks fifth in cancer mortality. [3] Although several therapeutic modalities for NHL including chemotherapy, radiation therapy, immunotherapy and radio-immunotherapy are in use, they have their own limitations for successful application in cancer therapy. An approach to combine different modalities of therapy adds on benefit by mitigating the limitations of each therapeutic modality. There is a recent report on the synergistic effect of chemotherapy combined with radio-immunotherapy. [4] In the present study, we have attempted to evaluate the extent of cell toxicity in Raji cells and the underlying mechanism when the cells were treated with doxorubicin in combination with 131 I-rituximab (rituximab radiolabeled with Iodine-131).

Doxorubicin is an anthracycline antibiotic and commonly used as a chemotherapeutic agent for a wide range of cancers. [5] There are pharmacological, and biological evidences supporting doxorubicin resistance in human tumor cells [6] and associated side effects of myocardiopathy. [7] The drug-resistance and associated side effects warrant the development of combinatorial modalities of therapy.

Rituximab, a chimeric monoclonal immunotherapeutic agent against CD20, has been approved by Food and Drug Administration in 1997. [8],[9] CD20 is a 33-37 kDa extracellular surface protein expressed during early B-cell development. [10] Most of the human B-cell-lineage malignancies express a large number of CD20 and, therefore, is considered to be an attractive, and one of the possible targets for the treatment of NHL. [11] Although rituximab is used for treatment of NHL patients, it is reported that it does induce resistance to therapy. [12],[13] It has been found that only 50% patients showed clinical response to rituximab. [3] To overcome the resistance and enhance the therapeutic efficacy in B-cell lymphoma, the combination of rituximab with various chemotherapeutic drugs (paclitaxel, gemcitabine, vinorelbine, doxorubicin and cisplatin) are being studied and reported to show an increase in cytotoxicity through various signaling pathways. [14],[15],[16],[17],[18],[19],[20],[21]

It has been shown that the combined modality of using rituximab and low-linear energy transfer irradiation (X-rays and γ-rays) enhanced apoptosis in NHL [22] and Burkitt lymphoma cell line. [23] Radio-immunotherapy using 131 I-rituximab for NHL, [24] relapsed or refractory indolent NHL patients [25],[26] and in Burkitt lymphoma cell line [27] is well reported. However, the effect on cell death induced by 131 I-rituximab in combination with doxorubicin and its underlying mechanism is not reported in detail. Herein, the author reports the study on cell toxicity in Raji cells treated with 131 I-rituximab in combination with doxorubicin. Furthermore, magnitude of apoptosis and alteration in mitogen-activated protein kinase (MAPK) signaling pathway were also evaluated in Raji cells treated with 131 I-rituximab in combination with doxorubicin.


 > Materials and methods Top


Materials

Chemicals and kits for assays were purchased from Sigma Chemical Inc. (St. Louis MO, USA) unless otherwise stated. Cleaved poly (ADP-ribose) polymerase (PARP) ELISA kit and "in situ Cell Death Detection ELISA kit" were purchased from Abcam, Tokyo, Japan and Roche Diagnostics GmbH (Indianapolis, IN, USA) respectively. 131 I was obtained from Radiochemicals Section of the erstwhile Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai, India. 131 I-rituximab was prepared as reported previously. [27]

Cell culture

Raji cells were obtained from National Center for Cell Sciences Pune, India. The cells were cultured in RPMI-1640 supplemented with 10% serum (Invitrogen Carlsbad, CA, USA), and antibiotic solution and incubated at 37°C in humidified incubator of 5% CO 2 atmosphere. Raji cells were treated at ~80% confluency.

Determination of the effect of doxorubicin on Raji cells

Raji cells (1 × 10 6 ) were plated in 24 well plates and treated with different concentration of doxorubicin (0.5-15μg/mL) for 6 h. At the end of incubation, cells were washed with phosphate buffered saline (PBS) and 1 × 10 3 cells were plated in 96 well plates which were further incubated for 12 and 24 h. Cells were subjected to 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, carried out according to protocol standardized for the study of radiolabeled molecules. [28] Briefly, after the incubation, 10 μL of MTT solution (5 mg/mL) was added in each well and incubated for 2 h. Formazan crystal was solubilized by addition of 100 μL of the solubilizing buffer (20% sodium dodecyl sulfate [SDS] in 50% dimethylformamide). The color obtained was quantified at 570 nm with reference to 630 nm in BioTek Universal Microplate Reader (BioTek, Winooski, VT, USA). Percent cell proliferation was calculated as the percentage of the ratio of optical density (OD) of treated and control samples.

Treatment of Raji cells with 131 I-rituximab and doxorubicin

Raji cells (1 × 10 6 ) were plated in 24 well plates and treated with doxorubicin (0.5, 1, 2 and 10 μg/mL) for 4 h. After that 1.85 MBq of 131 I-rituximab (or 5 μg/mL cold rituximab as control) was added for another 2 h. Hence, the total incubation of doxorubicin and 131 I-rituximab was for 6 and 2 h, respectively. The use of 1.85 MBq of 131 I-rituximab concentration was chosen from a previous study. [27] The cells were washed thrice with PBS at the end of a total incubation of 6 h, and further incubated for 12 h at 37°C. After 12 h, the cells were harvested to carry out further experiments.

Estimation of cell death in Raji cells treated with doxorubicin and 131 I-rituximab

Raji cells were treated with doxorubicin (0.5, 1, 2, and 10 μg/mL), rituximab and 131 I-rituximab either alone or in combination as mentioned above. Cells were washed with PBS and further incubated for 12 h. The cells were harvested and magnitudes of cell death in control and treated cells were determined by trypan blue dye. The cells were mixed with an equal volume of 0.4% w/v trypan blue dye. Living cells exclude the dye while dead cells take up the dye. The cells were counted using a hemocytometer and expressed as percent cells death. Percent cell death was calculated as the percentage of the ratio of dead and total cells.

Determination of apoptosis by DNA fragmentation study

DNA fragmentation study was carried out according to protocol described in the in situ Cell Death Detection ELISA kit plus. Briefly, Raji cells treated with doxorubicin and 131 I-rituximab were harvested. About 1 × 10 5 cells were lysed in lysis buffer for 30 min and centrifuged to 20,000 × g for 10 min. The supernatant was carefully transferred to new tubes. Diluted supernatant (20 μL of 1:10 v/v) was added into streptavidin coated microplate well. Anti-histone-biotin and anti DNA-peroxidase solutions were mixed prior to use in the ratio of 1:1 v/v and from which 80 μL of this solution was added to each well and incubated with gentle shaking for 2 h. Thereafter, the wells were washed thrice with buffer provided with the kit and incubated with substrate solution for 25 min. The absorbance was recorded at 405 nm. DNA fragmentation was expressed as enrichment factor (EF) which is the ratio of ODs of treated and control samples.

Study of apoptosis by estimation of poly (ADP-ribose) polymerase cleavage

Estimation of PARP cleavage was carried out according to the protocol described in cleaved PARP human ELISA kit. Briefly, doxorubicin and 131 I-rituximab treated Raji cells were harvested and lysed in cell lysis buffer for 45 min and centrifuged to 20,000 × g for 20 min. The supernatant protein was carefully transferred to new tubes and stored at −80°C until analysis. The protein in the supernatant was quantified by Bio-Rad Protein Assay (Bio-Rad lab Inc., Hercules CA, USA). Sixty microgram of supernatant protein was added in anti-PARP coated ELISA plate which was incubated for 2 h with gentle shaking. Thereafter, the wells were washed twice with buffer provided with the kit and incubated with detector antibody for 1 h. The wells were again washed twice and incubated with HRP labeled antibody for 1 h. The wells were finally washed thrice and incubated with substrate for 25 min. The reaction was stopped by the addition of 1N HCl and the color developed was quantified at 450 nm. PARP cleavage was expressed as EF which is the ratio of ODs of treated and control samples.

Study of protein expression, related to apoptotic cell death and mitogen-activated protein kinase pathways

Raji cells were harvested after above mentioned treatment and lysed in cell lysis buffer (10 mM Tris pH 7.4, 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 1 mM NaF, 20 mM Na 4 P 2 O 7 , 20 mM Na 3 VO 4 , 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 1 mM phenylmethylsulfonyl fluoride) and protease inhibitor cocktail. Protein concentration in samples was estimated by Bio-Rad Protein assay. Protein (40 μg) was loaded on 10% (for PARP) and 15% (for B-cell lymphoma-extra-large [bclxl]) SDS polyacrylamide gel electrophoresis and electrophoresis was carried out. Protein bands were transferred onto nitrocellulose membrane by electroblotting. The membrane was blocked by using 5% nonfat milk protein followed by incubation with primary antibodies for PARP, bclxl, MAPK pathways proteins (p44/42, p38 and its phosphoproteins) and beta-actin, for 1.5 h. The membrane was washed with tris buffer saline containing Tween-20 (0.1%) followed by treatment with secondary antibody. The membrane was incubated with SignalFire TM  ECL reagents (Cell Signaling Tech., Danvers, MA), followed by exposure to X-ray film (Amersham Hyperfilm ECL GE-healthcare, Buckinghamshire UK). X-ray film was processed and developed by Kodak developer and fixer solution. Densitometry of protein blot was carried out using UVIbandmap software (UVItech Limited, Cambridge, UK).

Statistical analysis

Unless mentioned, the results are mean ± standard deviation of at least three independent experiments where, P ≤ 0.05 are considered as significant.


 > Results Top


Effect of doxorubicin on cell proliferation of Raji cells

  MTT assay was carried out in Raji cells treated with increasing concentrations of doxorubicin (0.5-15 μg/mL) for 6 h. Percent cell proliferation observed at 12 h and 24 h was plotted against the doxorubicin concentration [Figure 1]. A dose-dependent decrease in proliferation was observed in Raji cells, which showed saturation at concentration of 8 μg/mL or above. A marginal difference in percent cell proliferation was observed between 12 and 24 h post incubation at higher concentration of doxorubicin (>8 μg/mL). However, no significant difference was observed at these incubation periods at lower concentrations of doxorubicin (<4 μg/mL).
Figure 1: Estimation of percent cell proliferation by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay of Raji cells treated with doxorubicin for 6 h

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Effect of doxorubicin and 131 I-rituximab on cell death of Raji cells

In order to investigate efficacy of doxorubicin combined with 131 I-rituximab, cell death was evaluated in Raji cells after treatment with 0.5, 1, 2 and 10 μg/mL of doxorubicin in combination of 1.85 MBq of 131 I-rituximab at 12 h [Figure 2]. Increased cell death was observed in Raji cells when treated with 131 I-rituximab in combination with doxorubicin compared to their respective controls of either doxorubicin or 131 I-rituximab. The magnitude of cell death in case of doxorubicin (2 μg/mL) in combination with 131 I-rituximab treated Raji cells was higher (~74%) compared to either doxorubicin (2 μg/mL, ~55%) or 131 I-rituximab (~22%) (P ≤ 0.05). 131 I-rituximab in combination with 10 μg/mL of doxorubicin induced the highest amount of cell death. However, only a marginal difference were observed in the case of 0.5 and 1 μg/mL of doxorubicin in combination of 131 I-rituximab.
Figure 2: Estimation of cell death in Raji cells treated with doxorubicin in combination with 131I-rituximab by trypan blue dye

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Effect of doxorubicin and 131 I-rituximab on induction of apoptotic DNA fragmentation in Raji cells

In order to investigate the underlying mechanism of cell death in Raji cells, after treatment with doxorubicin in combination of 131 I-rituximab, magnitude of apoptotic DNA fragmentation was estimated by ELISA method. The results showed that there is a significant (P ≤ 0.05) increase of EF confirming DNA fragmentation in the combined treatment of doxorubicin (2 μg/mL) and 131 I-rituximab (EF ~ 9) compared to the corresponding controls of doxorubicin (2 μg/mL, EF-4.2) and 131 I-rituximab (EF ~ 2.5) [Figure 3]a.. However, at higher concentration of doxorubicin (10 μg/mL), the combined treatment showed lesser DNA fragmentation (EF-~7) compared to the corresponding control of doxorubicin (10 μg/mL, EF ~ 8). These results are further expressed in terms of fold change in EF compared to their respective controls (either rituximab or doxorubicin) in [Figure 3]b. The figure showed maximum synergistic effect in terms of DNA fragmentation induced by combination at 2 μg/mL of doxorubicin with 131 I-rituximab compared to other combinations (0.5, 1 and 10 μg/mL of doxorubicin).
Figure 3: (a) Estimation of apoptotic DNA fragmentation of Raji cells treated with doxorubicin in combination with 131I-rituximab, (b) Apoptotic DNA fragmentation of Raji cells as a ratio of enrichment factor. (where 1: Phosphate buffered saline/control cells, 2: 1.85 MBq of 131I-rituximab /5 ìg of rituximab, 3: 0.5 ìg/mL of doxorubicin in combination with 1.85 MBq of 131I-rituximab/0.5 ìg/mL of doxorubicin, 4: 1 ìg/mL of doxorubicin in combination with 1.85 MBq of 131I-rituximab/1 ìg/mL of doxorubicin 5: 2 ìg/mL of doxorubicin in combination with 1.85 MBq of 131I-rituximab/2 ìg/mL of doxorubicin and 6: 10 ìg/mL of doxorubicin in combination with 1.85 MBq of 131I-rituximab/10 ìg/mL of doxorubicin.)

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Effect of doxorubicin and 131 I-rituximab on cleavage of poly (ADP-ribose) polymerase protein in Raji cells

This study was carried out to estimate the cleavage of PARP protein by ELISA method in control and treated Raji cells. Our results [Figure 4]a showed that there was a significant increase (P ≤ 0.05) of PARP cleavage in cells treated with doxorubicin (2 μg/mL) in combination with 131 I-rituximab (EF ~ 7.5) compared to their corresponding controls, that is, doxorubicin (2 μg/mL, EF ~ 3.45) and 131 I-rituximab (EF ~ 2.75). A marginal increase of PARP cleavage was also observed in cells treated with 0.5 and 1 μg/mL of doxorubicin in combination with 131 I-rituximab. The ratio of EF of the combined treatment to EF of individual controls [Figure 4]b, showed similar results.
Figure 4: (a) Estimation of poly(ADP-ribose) polymerase cleavage of Raji cells treated with doxorubicin in combination with 131I-rituximab, (b) poly(ADP-ribose) polymerase cleavage of Raji cells as a ratio of enrichment factor. (where 1: Cells treated in phosphate buffered saline/control cells, 2: Cells treated with 1.85 MBq of 131I-rituximab /5 ìg of rituximab, 3: Cells treated with 0.5 ìg/mL of doxorubicin in combination with 1.85 MBq of 131I-rituximab/0.5 ìg/mL of doxorubicin, 4: Cells treated with 1 ìg/mL of doxorubicin in combination with 1.85 MBq of 131I-rituximab/1 ìg/mL of doxorubicin and 5: Cells treated with 2 ìg/mL of doxorubicin in combination with 1.85 MBq of 131I-rituximab/2 ìg/mL of doxorubicin)

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Effect of doxorubicin and 131 I-rituximab on proteins involved in cell death and mitogen-activated protein kinase pathway

The results from trypan blue, DNA fragmentation and PARP cleavage assays showed increased efficacy of combined approach of doxorubicin and 131 I-rituximab. To further illustrate the underlying molecular players and signaling pathways, the regulation of bclxl in control and treated cells were studied. Additionally, the PARP cleavage observed by ELISA method was also verified by Western blotting [Figure 5]a. The results showed that the expression of anti-apoptotic protein (bclxl) was downregulated and cleaved PARP was higher in the cells treated with 131 I-rituximab in combination with doxorubicin (2 μg/mL) compared to the individual controls [Figure 5]a. Densitometry of bclxl and cleaved PARP proteins from Western blotting was carried out and expressed as a ratio of protein of interest to the corresponding beta-actin protein as a loading control [Figure 5]b. Maximum downregulation of bclxl and maximum cleavage of PARP protein was observed in the combined treatment compared to the individual controls.
Figure 5: (a) Expressions of apoptotic proteins in Raji cells treated with doxorubicin in combination with 131I-rituximab, (b) Ratio of Western blotting band intensity of apoptotic proteins with respect to the beta actin (loading control), (c) Expressions of mitogen-activated protein Kinases in Raji cells treated with doxorubicin in combination with 131I-rituximab, (d) Ratio of Western blotting band intensity of mitogen-activated protein kinases with respect to the beta actin (loading control)

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Western blotting was also carried out for MAPK pathway proteins, that is, p44/42, p38 and their phosphorylated form, in control and treated cells. An increase in expression of p38 proteins was observed in cells treated in combination compared to the individual controls, while no change in expression was observed for p44/42 (except marginal decrease in rituximab/ 131 I-rituximab controls, P ≤ 0.05) [Figure 5]c and d. However, strong phosphorylation of p38 was observed in 131 I-rituximab treated cells while phosphorylation of p38 was lower in cells treated with 131 I-rituximab in combination of doxorubicin. In the case of phospho p44/42 proteins [Figure 5]c and d, the phosphorylation was lower in doxorubicin and combined treatment. Densitometry of p38, p44/42, phospho p38 and phospho p44/42 were analyzed and expressed as a ratio of protein of interest to the corresponding beta-actin protein as a loading control [Figure 5]d. It was found that almost complete downregulation of phospho p44/42 was observed in the combinatorial treatment. Similarly, phospho p38 was downregulated in the combined treatment compared to the 131 I-rituximab controls.


 > Discussion Top


The dosages of doxorubicin administered are 60-75 mg/m 2 or 1.2-2.4 mg/kg of body weight for cancer chemotherapy. However, these dosages cause toxicity to cardiomyocytes [7] and chemo-resistance to the therapy. [6] To elucidate the mechanism underlying the cytotoxicity in Raji cells after doxorubicin and 131 I-rituximab treatment, we have estimated cell death, apoptosis by DNA fragmentation and PARP cleavage and regulation of MAPK signaling pathways. Rituximab is known to cause cytotoxicity in tumor cells involving various cellular signaling pathways. [3],[ 21] Additional killing of lymphoma cells by 131 I-rituximab will be due to associated high energy beta radiation (0.6 MeV of 131 I). The enhanced magnitudes of damage are due to the localization of radio-isotope very close to cellular targets at membrane and cytoplasm level. [27]

The higher magnitude of cell death measured by trypan blue dye was observed after treatment of doxorubicin (10 μg/mL) in combination with 131 I-rituximab, compared to other combined treatments of lower concentration of doxorubicin and 131 I-rituximab. However, the extent of DNA fragmentation was more in case of treatment of doxorubicin (2 μg/mL) in combination with 131 I-rituximab. At higher concentration of doxorubicin (10 μg/mL), the combination of 131 I-rituximab resulted in lower DNA fragmentation in terms of EF values.The combinatorial treatment of doxorubicin at higher concentration would result in higher apoptosis and thus there is an increase in the number of cell lysis at the advanced stage of cell death. The fragmented DNA from these lysed cells would be lost in the supernatant while harvesting the cells for DNA fragmentation analysis by ELISA. Similarly, the effects of combinatorial treatments of 0.5 and 1 μg/mL of doxorubicin were lower than that of 2 μg/mL. The observed cleaved PARP protein determined by Western blotting was similar to PARP cleavage by ELISA method. This apoptotic cell death was higher in the combinatorial treatment as shown in DNA fragmentation, which was further correlated with downregulation of bclxl and PARP cleavage studied by Western blotting. These results are in agreement with observation of enhanced cytotoxicity of cold rituximab with drugs (cisplatin, adriamycin, vinblastine, 5-fluorouracil and paclitaxel) in the NHL B-cell line. [14],[15],[16],[17],[18],[19],[20],[21] In general, p38 are poorly activated by mitogens but strongly activated by inflammatory cytokines and a wide variety of cellular stress inducers and its activation occurs through phosphorylation. After activation, these cytosolic proteins get translocated to the nucleus and activate numerous proteins and/or transcription factors. [29],[30] So the increase in expression of p38 protein in the combination of 2 μg/mL of doxorubicin with the 131 I-rituximab might be due to the radiation stress. The higher phosphorylation of p38 in 131 I-rituximab treated cells and relatively lower phosphorylation was observed in combinatorial treatments, which is in agreement with previous studies showing decrease in p38 phosphorylation when cold rituximab was combined with drugs in B-cell chronic lymphocytic leukemia cell line [31],[32] and 2F7 B NHL cell line. [33],[34] However, the mechanism still needs to be investigated. Conversely to p38, p44/42 (also known as ERK1/2) is a key transducer signal and is often activated by mitogens during proliferation. Its activation occurs through phosphorylation of threonine and tyrosine. The increase in cytotoxicity in case of 131 I-rituximab in combination of doxorubicin may be due to a decrease in phosphorylation of p44/42. On downstream signaling, p44/42 is known to inhibit bclxl [19] leading to cell death, which may explain the association of the signaling pathway in enhanced killing of Raji cells treated with 131 I-rituximab in combination with doxorubicin. Our results suggest that doxorubicin has the potential to sensitize 131 I-rituximab induced cell death in Raji cells, which has relevance in the radio-nuclide therapy of NHL. However, validation of the combinatorial approach in other lymphoma cell lines and suitable animal model is required to translate the research to clinics.


 > Acknowledgments Top


The authors are thankful to their colleagues in Radiochemical Section and Radiation Sources Section, Isotope Applications and Radiopharmaceuticals Division (IA and RPhD), Bhabha Atomic Research Centre, Mumbai, India for providing the I-131 used for radioiodination in the study. The authors gratefully acknowledge the continuous support of Dr Gursharan Singh, Associate Director, Radiochemistry and Isotope Group and Head IA and RPhD BARC, Mumbai, India.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]



 

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