Anticancer effects of resveratrol in canine hemangiosarcoma cell lines

Hemangiosarcoma (HSA) is a highly malignant tumour with aggressive biological behaviour. HSAs are more common in dogs than other domestic animals. The median survival time of dogs with HSA remains short, even with chemotherapy and surgery. Therefore, there is a critical need to improve the adjuvant chemotherapeutic regimens to improve clinical outcomes in dogs with HSA. Resveratrol has been shown to possess strong anti‐proliferative and/or pro‐apoptotic properties in human cancer cell lines. Nevertheless, the potential anticancer effects of resveratrol have not been reported in canine HSAs. The objective of this study is to determine the growth inhibitory effects of resveratrol in HSA cells when used alone or in combination with doxorubicin, a commonly used chemotherapeutic agent. Frog and DD‐1 canine HSA cell lines were treated with varying concentrations of resveratrol with and without doxorubicin. Cell viability was measured by the MTT assay. The expression of apoptotic proteins, activation of p38 mitogen‐activated protein kinase (MAPK), AMP‐activated protein kinase (AMPK) and extracellular signal‐regulated kinase 1/2 (ERK1/2) were assessed by western blotting. Similar to human cancer cell lines, resveratrol markedly inhibited the growth and induced apoptosis in both HSA cell lines. Mechanistically, resveratrol activated p38 MAPK, but did not affect the AMPK or the ERK1/2 pathways. Additional experiments showed that resveratrol augmented the growth‐inhibitory and apoptotic effects of doxorubicin in both HSA cell lines. These findings suggest that resveratrol has pro‐apoptotic effects in canine HSA cells; therefore, its use as a potential adjunct therapy in canine HSA patients warrants further investigation.     

Prognostic significance of clinical presentation,induction and rescue treatment in 42 cases of canine centroblastic diffuse large B‐cell multicentric lymphoma in the United Kingdom

Canine lymphoma is a heterogeneous group of diseases and many previous studies have evaluated the response of a mixed population of lymphoma cases to one specific treatment protocol. The aim of this retrospective study was to describe the outcome and prognostic factors in 42 cases of multicentric centroblastic diffuse large B‐cell lymphoma treated with either a COP‐type (35%) or CHOP‐type (64%) induction chemotherapy. The objective response rate to induction therapy was 94%; entire dogs had a greater rate of complete vs partial remissions than neutered dogs (P = .017). Median progression‐free survival for the first remission (PFS1) was 182 days; absence of anaemia at diagnosis (P = .002) and pretreatment neutrophil:lymphocyte ratio (NLR) below 9.44 (P = .015) were independently predictive of longer PFS1. Fifty‐eight percent of dogs received rescue protocols with an objective response rate of 81%; 31% of dogs received further rescue protocols (up to a total of 5) and the median number of protocols administered were 2. Median overall survival (OS) was 322 days, the 1‐year survival rate was 38% and the 2‐year survival rate was 9%. Lymphocyte:monocyte ratio above 1.43 (P = .031), NLR below 11.44 (P = .009), the combination of induction and rescue therapy (P = .030) and the total number of doxorubicin doses used (P = .002) were independently predictive of longer OS. Use of a COP‐type protocol induction compared with CHOP did not undermine OS providing doxorubicin was used as rescue therapy.     

茶多糖对阿霉素抑制肺癌A549细胞增殖作用的影响

肺癌是全世界目前发病率和死亡率最高的癌症之一,当前治疗恶性肿瘤的主要手段之一是化疗,阿霉素是临床常用的化疗药物,然而该药物毒性较大,长期使用可发生剂量依赖性的不可逆的心肌病变、骨髓抑制等,同时其多药耐药性的存在也使它在临床应用受到一定限制。为了减少阿霉素的毒副作用,通过体外培养人肺癌A549细胞,将茶叶提取物茶多糖与阿霉素联用,加入A549细胞,24 h后以噻唑蓝(MTT)法检测细胞存活率。结果表明,当阿霉素质量浓度为3 mg·L~(-1)时,对肺癌A549细胞的抑制效果最明显;不同浓度茶多糖与1、2、3 mg·L~(-1)阿霉素联用,以2 mg·L~(-1)阿霉素与6 mg·L~(-1)茶多糖联用时对肺癌A549细胞的抑制效果最明显,且优于单独使用3 mg·L~(-1)阿霉素的效果。阿霉素可诱导A549细胞凋亡,茶多糖与阿霉素联用可减少阿霉素的使用剂量,增强阿霉素对肺癌A549细胞的增殖抑制作用。     

Immortalization and characterization of a new canine mammary tumour cell line FR37‐CMT

Here we describe the establishment of a new canine mammary tumour (CMT) cell line, FR37‐CMT that does not show dependence on female hormonal signaling to induce tumour xenografts in NOD‐SCID mice. FR37‐CMT cell line has a stellate or fusiform shape, displays the ability to reorganize the collagen matrix, expresses vimentin, CD44 and shows the loss of E‐cadherin which is considered a fundamental event in epithelial to mesenchymal transition (EMT). The up‐regulation of ZEB1, the detection of phosphorylated ERK1/2 and the downregulation of DICER1 and miR‐200c are also in accordance with the mesenchymal characteristics of FR37‐CMT cell line. FR37‐CMT shows a higher resistance to cisplatin (IC₅₀>50 µM) and to doxorubicin (IC₅₀>5.3 µM) compared with other CMT cell lines. These results support the use of FR37‐CMT as a new CMT model that may assist the understanding of the molecular mechanisms underlying EMT, CMT drug resistance, fostering the development of novel therapies targeting CMT.     

CONCOMITANT LIPOSOMAL DOXORUBICIN AND DAILY PALLIATIVE RADIOTHERAPY IN ADVANCED FELINE SOFT TISSUE SARCOMAS

Local recurrence of feline soft tissue sarcomas is common despite aggressive treatment. Liposomal doxorubicin might serve as a depot radiosensitizer if administered concomitantly with daily radiotherapy and thus improve tumor control. In this pilot study, the feasibility of concomitant liposomal radiochemotherapy was evaluated in a palliative setting in 10 cats with advanced soft tissue sarcomas. Cats were treated with median number of 5 (range 5–7) daily fractions of radiotherapy and a median total dose of 20 Gy (range 20–31.5 Gy). One dose of liposomal doxorubicin was administered at the beginning of radiotherapy. Seven cats received further free or liposomal doxorubicin after completion of the liposomal doxorubicin/radiation protocol. Seven of the treated 10 cats (70%) achieved a partial (n=5) or complete (n=2) response with a median response duration of 237 days. The median progression free interval in all 10 cats was 117 days and the median overall survival time was 324 days. Concomitant liposomal radiochemotherapy was tolerated well in nine cats, one cat experienced temporary anorexia. Although the number of patients is too small to make definitive conclusions, results appear promising enough to investigate the role of liposomal doxorubicin as a radiosensitizer further.     

Efficacy of doxorubicin‐based chemotherapy for non‐resectable canine subcutaneous haemangiosarcoma

Eighteen dogs with measurable subcutaneous haemangiosarcoma (SQHSA) were treated with doxorubicin‐based chemotherapy. Response assessment was evaluated and compared using World Health Organization (WHO), Response Evaluation Criteria in Solid Tumours (RECIST) and tumour volume criteria. The overall response rate for all dogs was 38.8% using WHO criteria, 38.8% using RECIST criteria and 44% using tumour volume criteria. One dog had a complete response. The median response duration for all dogs was 53 days (range 13–190 days). Four dogs had complete surgical excision after neoadjuvant chemotherapy. The median progression‐free interval for dogs with complete surgical excision after neoadjuvant chemotherapy was significantly longer than those not having surgical excision (207 days versus 83 days, respectively) (P = 0.003). No significant difference in metastasis‐free interval or survival time was found between the groups. Doxorubicin‐based chemotherapy appears to be effective for non‐resectable canine SQHSA, although the response duration is relatively short.     

Evaluation of bone toxicity in various bones of aged rats

The aim of the present study was to provide a method for evaluating bone toxicity induced by drugs in various bones in aged rats. Male Crl:CD (SD) rats at 46 weeks of age were administered 15 mg/m(2) body surface area of doxorubicin, which effects the growth plate in weanling rats, weekly for 9 weeks by intravenous injection, and the femur, sternum, humerus and tibia were examined histopathologically. In the doxorubicin-treated group, thinning of the growth plate was remarkably observed in the proximal tibia and humerus; however, these changes were not observed in other regions. In addition, the osteoclast number per bone perimeter in the proximal tibia was significantly higher than others in control aged rat. Thus, recognizing the various histological reactions related to the time of epiphyseal closure is important for evaluating bone toxicity in aged rats.     

A study on the mechanism of doxorubicin promoting IL-1β and collagen type I secretion in cardiac fibroblasts

AIM To observe the effect of adriamycin/doxorubicin （DOX） on the production of inflammatory cytokines and collagen in cardiac fibroblasts and its mechanism. METHODS Neonatal SD rat cardiac fibroblasts were isolated, cultured, and identified by immunofluorescence staining with monoclonal antibodies against vimentin observed under a confocal laser-scanning microscope. The Cell Counting Kit-8 assay was used to detect the toxicity of DOX on cardiac fibroblasts, and flow cytometry with annexin V-FITC/PI double staining was used to detect apoptosis. ELISA was used to detect the release of inflammatory factors in the supernatant of cultured cells. Immunofluorescence labeling assay was used to detected α-smooth muscle actin （α-SMA） expression and mitochondrial reactive oxygen species （mROS） in the cells. Western blot was used to detect the expression of nucleotide-binding oligomerization domain-like receptor protein 3 （NLRP3） inflammasome-related proteins in cardiac fibroblasts. RESULTS （1） Compared with the control group, DOX inhibited the proliferation of cardiac fibroblasts （P<0.05）, but had no significant effect on apoptosis （P>0.05）. （2） Treatment with DOX promotes the release of proinflammatory factors interleukin-1β （IL-1β） and IL-6 in cardiac fibroblasts （P<0.05）. （3） The expression of α-SMA, collagen type I and transforming growth factor-β in DOX treatment group increased significantly compared with control group （P<0.05）. （4） Compared with the control group, the levels of mROS, cellular NLRP3 and cleaved caspase-1 in cardiac fibroblasts increased significantly after DOX treatment. CONCLUSION Doxorubicin promotes cardiac fibroblasts to secrete IL-1β and collagen type I by promoting mROS production and activating NLRP3 inflammasome.     

Doxorubicin area under the curve is an important predictor of neutropenia in dogs with naturally occurring cancers

Doxorubicin (DOX) area‐under‐the‐curve (AUC) was calculated for 40 dogs with spontaneously occurring cancers using a previously validated limited‐sampling approach. All dogs were administered a dose of 30 mg/m² by intravenous infusion and serum samples were collected at 5, 45 and 60 minutes post‐infusion. DOX and its major metabolite, doxorubicinol (doxol), were quantified in serum samples using high‐performance liquid chromatography tandem‐mass spectrometry. Wide interpatient variability was observed in the predicted DOX AUC with a coefficient of variation of 34%. A significant relationship was found between DOX AUC and absolute white blood cell count (P = 0.003), absolute neutrophil count (ANC; P = 0.002) and surviving fraction of neutrophils (P = 0.03) approximately 1 week after dosing (nadir). No changes in other hematologic parameters (red blood cells, platelets, lymphocytes, haemoglobin) were found to correlate with DOX AUC. The absolute dose (mg) and the dose per unit body weight (mg/kg) were not significantly correlated with nadir ANC. No relationships were found between maximum serum doxol concentration and myelosuppression. Baseline ANC was also significantly correlated to nadir ANC and a model was constructed using baseline ANC and DOX AUC that significantly described the nadir ANC. These findings demonstrate the important relationship between systemic DOX exposure and degree of neutropenia in dogs, and suggest a potential for individualized, pharmacokinetically‐guided DOX dosing in dogs.     

Timely adjuvant chemotherapy improves outcome in dogs with non-metastatic splenic hemangiosarcoma undergoing splenectomy

Timely delivery of adjuvant chemotherapy has been shown to be advantageous in many human cancers and canine osteosarcoma. Adjuvant chemotherapy has been shown to improve outcome for canine splenic hemangiosarcoma. The aim of this retrospective study was to investigate whether timely adjuvant chemotherapy administration resulted in better outcome in dogs with non-metastatic splenic hemangiosarcoma undergoing splenectomy. Medical records were searched for dogs with non-metastatic, splenic hemangiosarcoma that received splenectomy and adjuvant chemotherapy. The number of days from surgery to the first chemotherapy dose (StoC) was evaluated to identify the cut-off value associated with the best survival advantage. StoC and other possible prognostic factors were tested for influence on time to metastasis (TTM) and overall survival (OS). Seventy dogs were included. Median StoC was 20 days (range: 4–70). The time interval associated with the greatest survival benefit was 21 days. Median TTM and OS of dogs with StoC ≤ 21 days were significantly longer than those with StoC >21 days (TTM: 163 vs. 118 days, p = .001; OS: 238 vs. 146 days, p < .001). On multivariable analysis, StoC >21 days was the only variable significantly associated with increased risk of tumour progression (HR 2.1, p = .010) and death (HR 2.3; p = .008). Starting adjuvant chemotherapy within 21 days of surgery may be associated with a survival benefit in dogs with non-metastatic splenic hemangiosarcoma, possibly due to the early targeting of newly recruited metastatic cells after surgery.