Celecoxib Prevents Doxorubicin-Induced Multidrug Resistance in Canine and Mouse Lymphoma Cell Lines

Background: Treatment of malignancies is still a major challenge in human and canine cancer, mostly due to the emergence of multidrug resistance (MDR). One of the main contributors of MDR is the overexpression P-glycoprotein (Pgp), which recognizes and extrudes various chemotherapeutics from cancer cells. Methods: To study mechanisms underlying the development of drug resistance, we established an in vitro treatment protocol to rapidly induce Pgp-mediated MDR in cancer cells. Based on a clinical observation showing that a 33-day-long, unplanned drug holiday can reverse the MDR phenotype of a canine diffuse large B-cell lymphoma patient, our aim was to use the established assay to prevent the emergence of drug resistance in the early stages of treatment. Results: We showed that an in vitro drug holiday results in the decrease of Pgp expression in MDR cell lines. Surprisingly, celecoxib, a known COX-2 inhibitor, prevented the emergence of drug-induced MDR in murine and canine lymphoma cell lines. Conclusions: Our findings suggest that celecoxib could significantly improve the efficiency of chemotherapy by preventing the development of MDR in B-cell lymphoma.

. Immunohistochemical findings of the two patients.

Patient 1 Markers Results
Immunohistochemistry CD79a Diffuse positivity. CD3 Tumor cells do not express the antigen. Ki67 Average 30-40% positivity. Diagnosis Large cell immunoblastic lymphoma Patient 2 Immunohistochemistry CD79a Diffuse positivity. CD3 Expression cells are scattered in the tumor area. Ki67 Average 60% positivity. Diagnosis Diffuse large B-cell lymphoma Two canine patients were diagnosed with B-cell lymphoma according to the immunohistochemistry.

Supplementary Materials 1. Cytology Reports
Centroblastic type (Kiel) monomorphic subtype composed of more than 60% centroblasts, which are large cells with scant basophilic cytoplasm, a round nucleus, fine chromatin pattern, and 2-4 basophilic prominent nucleoli located in the margin [2]. They are 10-30 μm in diameter, and the nucleus is less completely heterochromatic than that of in small lymphocyte (they sometimes referred to as large lymphocytes or lymphoblasts) [3]. The cytoplasm is pale blue and more abundant than in small lymphocytes. Their nuclei are 1.5-3 times the size of a red blood cells (RBC) or larger to up to 4 times the size of an RBC. Some nuclei have one moderately large incision (or cleaved). The nuclei of them have a fine diffuse and light chromatin pattern. Nucleoli are prominent, and can be well visible with characteristic margins, and even multiple and/or prominent. The cytoplasm is abundant and often basophilic and may completely encircle the nucleus. Occasionally pale Golgi zone is seen besides the nucleus, at the incision of it. Mitotic figures were estimated by looking at 5 cellular fields under 40x power. In this case it was moderate: 2-3 mitotic figures [4].

Supplementary Methods 1. Method of Cytology Sampling
Criteria for the involvement of bone marrow by lymphoma were: (1) the presence of > 20% lymphocytes in the sample and/or (2) the presence of large/atypical lymphocytes, even if the proportion was lower than 20% of all nucleated cells (ANC) [5].
The lymph node and bone marrow samples were taken under general anaesthesia. The dogs were anaesthetized (propofol /AstraZeneca Co., Cambridge, UK/ 5 mg/BW kg iv, isoflurane/Abbott Ltd., Budapest, Hungary/ 1.5-2.5 V/V%, fentanyl/Gedeon Richter Plc., Budapest, Hungary/constant rate infusion 0.01 to 0.04 mg/BWkg/hr) and an enlarged lymph node was excised for routine histological and immunohistochemical examination. Bone marrow aspirates were taken for cytological analysis by using a Jamshidi needle from the iliac crest (crista iliaca externa). The aspirates were smeared and stained with a staining kit (Quick panoptic staining kit: Reagens Ltd., Budapest, Hungary) for cytological evaluation.
Sternal recumbency was used for the wing of the ilium Once the patient was sedated with the above protocol, a BMA was performed using a standard technique [6]. A 2.5cm × 2.5cm area of the skin was shaved, cleansed, and disinfected with chlorhexidine. Once the site was prepped and local anaesthesia (1-2 mL of lidocaine 2%) injected into the skin, subcutaneous tissues and on the periosteum of the bone, a small nick was made in the skin with a sterile #11 scalpel blade. The BM needle was angled slightly medially and parallel to the wing of the lium. A 15-gauge BM needle (with the stylet locked in place) was then firmly pushed through the subcutaneous tissues and the dense outer layer of the bone into the marrow. cavity. Once the needle was in contact with the surface of the bone, it was rotated into the bone in a clockwise/counter-clockwise motion. The stylet was then removed, and a 10-12 mL syringe containing 50 μL of 10% EDTA was attached to the needle, and vigorous suction was applied to withdraw a small amount of liquid marrow material into the syringe. The needle and syringe were then removed en bloc from the patient through application of firm traction to the needle. Immediately after collection of the sample, blood-contaminated BM was directly applied to glass slides (direct smears). A second slide was placed on top of the sample to