Comparison of the anti-cancer activity of 5-aminolevulinic acid-mediated photodynamic therapy after continuous wave and pulse irradiation in different histological types of canine mammary sarcoma tumors.

Canine mammary sarcoma tumors (CMST) are the most aggressive tumors with poor prognosis in dogs. Due to inadequate treatment options for CMST, recent studies have focused on alternative treatment strategies. We previously determined the optimized protocol of 5-ALA-based photodynamic therapy (PDT) in canine liposarcoma. However, its molecular mechanisms in the treatment of different histological types of CMST remain unclear.In this context, we, for the first time, assessed 5-aminolevulinic acid (5-ALA)-PDT-mediated anti-cancer activity and its molecular mechanism after continuous wave (CW) and pulse radiation (PR) on three different histological types (liposarcoma, chondrosarcoma, and osteosarcoma) of CMST cells by WST-1, Annexin V, ROS, acridine orange/propidium iodide staining, RT-PCR, and western blot analysis.Our findings showed that 5-ALA/PDT significantly suppressed the proliferation of CMST cells (p < 0.01) and induced apoptosis via increased ROS level and overexpression of Caspase-9 and Caspase-3 mRNA and cleaved protein levels in especially liposarcoma and chondrosarcoma cells following CW and PR irradiation at 9 J/cm2. However, the response of CMST cells to 5-ALA was different upon CW and PR irradiation due to differences in their origin.Collectively, our findings provided the first evidence that 5-ALA-based PDT could be used as an alternative treatment strategy, especially liposarcoma and chondrosarcoma. However, further in vitro and in vivo studies are required to elucidate the underlying molecular mechanism of the efficacy of 5-ALA in CMST cells at the molecular level.

Evidence for heterogeneity in response to treatment in mammary tumors of dogs as happens in humans.

Tumors are formed by various clones developed over a long time. This gives rise to a heterogeneous nature. This heterogeneity is the hardest challenge in the treatment of cancers because it is the main reason for drug resistance. This is a well-known fact in human cancer. Therefore, we have reasoned that if the tumor heterogeneity in canine mammary gland tumors (CMGTs) could be shown by an ex vivo assay, which will be used first time in veterinary oncology practice, this could be used further in clinics. To achieve this, twenty-six patients were included in the study. Tumor tissues were obtained from animals during routine surgery. Tumor cells were isolated and seeded ex vivo. The cells were exposed to anticancer drugs that are clinically used. Seven days after the treatment, chemosensitivity has luminometrically been assayed by ATP-tumor chemosensitivity assay (ATP-TCA). It has clearly been shown that all the tumor tissues have responded to treatment differently, implying that heterogeneity exists in mammary tumors. There has also been found that there was a weak to moderate statistically significant correlation between tumor size and drug index. However, there has been no correlation between drug index and metastasis to lymph nodes. Hyperplasic areas had relatively higher PCNA values. The results of our study demonstrate the heterogeneity in responses to in vitro drugs. Clinical trials based on test results and follow-up studies with large numbers of animals are needed to prove that such chemotherapeutic activity assessment tests can be clinically useful in predicting drug responses in CMGTs.

Efficacy of 5-aminolevulinic acid-based photodynamic therapy in different subtypes of canine mammary gland cancer cells.

Canine mammary gland tumors (CMGTs) are heterogeneous disease and subclassified [sarcomas (S), carcinomas (C), and carcinosarcomas (CS)] according to histopathological differentiation. Photodynamic therapy (PDT) is a promising treatment strategy based on the use of a photosensitizer (PS) activated by light. However, the therapeutic potential of PDT in the treatment of CMGTs has not been investigated, yet. Therefore, the aim of this study was to determine the in vitro protocol of 5-ALA-based-PDT for the treatment of three subtypes of CMGTs, for the first time. The intracellular PpIX florescence intensity was measured for 5-ALA (0.5 and 1 mM). After irradiation with different light doses (6, 9, 12, 18, and 24 J/cm2) for two different modes [continuous wave (CW) and pulse radiation (PR)], the cytotoxic effects of 5-ALA (0.5 and 1 mM) on the subtypes (C, S, and CS) of CMGTs were analyzed by WST-1. Finally, the optimal PDT treatment protocol was validated through Annexin V and AO/EtBr staining. Our results showed that 1 mM 5-ALA for 4-h incubation was the best treatment condition in all subtypes of CMGTs due to higher intracellular PpIX level. After irradiation with different light doses, PR mode was more effective in S primary cells at 9 J/cm2. However, a significant decrease in the viability of C and CS cells was detected at 12 /cm2 in CW mode (p < 0.05). Additionally, 1 mM 5-ALA induced apoptotic cell death in each subtype of CMGTs. Our preliminary findings suggest that (i) each subtype of CMGTs differentially responds to PDT and (ii) the light dose and mode could play an important role in the effective PDT treatment. However, further studies are needed to investigate the role of the different light sources and PDT-based apoptotic cell death in CMGTs cells.

Evaluation of changes in Doppler ultrasonography indices and levels of maternal serum angiogenic factors throughout pregnancy in ewes.

We evaluated the changes in the pulsatility index (PI) and resistance index (RI) of fetal and maternal vessels and maternal serum angiogenic factors throughout pregnancy in 20 Kivircik ewes. They were examined for pregnancy detection on Day 30 after mating. The PI and RI during the pregnancies were followed using Doppler ultrasonography (USG) of the uterine artery, umbilical artery, fetal aorta, and fetal vena cava. Doppler USG was performed every 15 days beginning from the 40th day after mating. Blood samples were collected every 15 days starting from the day of mating. Nitric oxide (NO), vascular endothelial growth factor, basic fibroblast growth factor, and angiopoietin-1 and -2 levels were measured in maternal serum. A gradual decline was observed in the PI and RI of the umbilical artery beginning from Day 75 of pregnancy. Similarly, the RI of the uterine artery decreased on Day 135. Embryonic resorption was detected in two ewes with an increased PI and RI in the uterine artery. A significant and gradual decrease in the maternal serum vascular endothelial growth factor level was observed throughout pregnancy. The maternal serum NO level increased beginning from Day 135 of pregnancy. The results of this study illustrate the progressive changes in the Doppler USG findings of fetomaternal vessels and maternal serum angiogenic factors that occur throughout ewe pregnancy. The umbilical and uterine artery Doppler USG findings and maternal serum NO concentration may be important parameters for evaluation of the course of pregnancy in ewes. The results of this study should be compared with those of further studies that include compromised pregnancies and nonpregnant ewes.

Follicular and steroid hormone changes in Arabian mares in the postpartum period.

In this study, the aim has been to characterize the profiles of progesterone, estradiol and follicular changes during postpartum period from parturition to first postpartum ovulation. Jugular venous blood was taken daily for progesterone and estradiol assay until first postpartum ovulation. The diameters of the follicles of the ovaries were recorded. The mean progesterone and estrogen concentrations markedly decreased (p<0.05) during the 48h after parturition. No correlation was observed between estradiol concentrations and the diameter of the largest follicle whereas a negative correlation was present between the number of follicles smaller than 20mm and the number of follicles larger than 20mm on the third and fourth days after postpartum.

The effect of oxytocin and PGF2alpha on the uterine involution and pregnancy rates in postpartum Arabian mares.

In this study, the effects of oxytocin and an analog of prostaglandin (cloprostenol) on the uterine involution and pregnancy rates were investigated. Mares received 3 ml of 0.9% NaCl in Group C (n=10), 30 IU/mare of oxytocin in Group O (n=10) and 250 microg/mare of cloprostenol in Group P (n=10) within 12h after parturition. The gravid uterine horn's cross-sectional diameter was measured by ultrasonography. The mean uterine diameters did not differ significantly between the treatment (O and P) and the control (C) groups (p>0.05). The difference between the postpartum ovulation periods (Group C: 12.6+/-0.72 days, Group O: 15+/-1.33 days, Group P: 14.6+/-1.11 days), the pregnancy rates at foal heat (Group C: 60%, Group O: 60%, Group P: 80%) and the embryonic death rates at foal heat (Group C: 33.3%, Group O: 16%, Group P: 25%) were not found to be statistically significant between the treatment and the control groups. The mean progesterone concentrations were similar in all groups and decreased continuously from parturition to until foal heat (Group C: from 2.43+/-0.24 to 0.66 ng/ml, Group O: from 3.07+/-0.6 to 0.27+/-0.27 ng/ml and Group P: from 2.8+/-0.44 to 0 ng/ml) (p>0.05). In conclusion, it was decided that the oxytocin and PGF2alpha treatments performed on the mares with the purpose of stimulating involution had no effect on the duration of parturition-first ovulation, the shrinkage of the uterus diameter, the pregnancy and embryonic death rates.