Enhancing radiosensitisation of BRCA2-proficient and BRCA2-deficient cell lines with hyperthermia and PARP1-i.

Poly(ADP-ribose)polymerase1 (PARP1) is an important enzyme in regulating DNA replication. Inhibition of PARP1 can lead to collapsed DNA forks which subsequently causes genomic instability, making DNA more susceptible in developing fatal DNA double strand breaks. PARP1-induced DNA damage is generally repaired by homologous recombination (HR), in which BRCA2 proteins are essential. Therefore, BRCA2-deficient tumour cells are susceptible to treatment with PARP1-inhibitors (PARP1-i). Recently, BRCA2 was shown to be down-regulated by hyperthermia (HT) temporarily, and this consequently inactivated HR for several hours. In this study, we investigated whether HT exclusively interferes with HR by analysing thermal radiosensitisation of BRCA2-proficient and deficient cells. After elucidating the equitoxicity of PARP1-i on BRCA2-proficient and deficient cells, we studied the cell survival, apoptosis, DNA damage (γ-H2AX foci and comet assay) and cell cycle distribution after different treatments. PARP1-i sensitivity strongly depends on the BRCA2 status. BRCA2-proficient and deficient cells are radiosensitised by HT, indicating that HT does not exclusively act by inhibition of HR. In all cell lines, the addition of HT to radiotherapy and PARP1-i resulted in the lowest cell survival, the highest levels of DNA damage and apoptotic levels compared to duo-modality treatments. Concluding, HT not only inhibits HR, but also has the capability of radiosensitising BRCA2-deficient cells. Thus, in case of BRCA2-mutation carriers, combining HT with PARP1-i may boost the treatment efficacy. This combination therapy would be effective for all patients with PARP1-i regardless of their BRCA status.

Dead or alive? Autofluorescence distinguishes heat-fixed from viable cells.

PURPOSE: A proof-of-concept study to evaluate a new autofluorescence method to differentiate necrotic thermally fixed cells from viable tissue following thermal ablation. METHODS: A conductive interstitial thermal therapy (CITT) device was used to ablate swine mammary tissue and rabbit VX-2 carcinomas in vivo. The ablated regions and 10-mm margins were resected 24 h following treatment, embedded in HistOmer and sectioned at 3 mm. The fresh sections were evaluated for gross viability with triphenyl tetrazolium chloride, 1 h post-resection. Representative non-viable and viable areas were then processed and embedded into paraffin, and sectioned at 5 microm. Standard H&E staining and proliferating cell nuclear antigen (PCNA) immunohistochemistry were compared against autofluorescence intensity, at 488-nm wavelength, for cellular viability. RESULTS: Heat-fixed cells in non-viable regions exhibit increased autofluorescence intensity compared to viable tissue (area under receiver operating characteristics (ROC) curve = 0.96; Mann-Whitney P < 0.0001). An autofluorescence intensity-based classification rule achieved 92% sensitivity with 100% specificity for distinguishing non-viable from viable samples. In contrast, PCNA staining did not reliably distinguish heat-fixed, dead cells from viable cells. CONCLUSIONS: Examination of H&E-stained sections using autofluorescence intensity-based classification is a reliable and readily available method to accurately identify heat-fixed cells in ablated surgical margins.

The change of proliferating cell nuclear antigen and apoptosis of the MM46 mammary cancer cells of the mouse after single high-dose irradiation.

The effects of intraoperative radiotherapy on tumor cells were elucidated by immunohistochemical examination of changes in the level of proliferating cell nuclear antigen. In addition, the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method was used to examine the level of apoptosis in mouse MM46 tumor cells after a single high dose of irradiation (30 Gy, 6 MeV). A significant decrease in the number of tumor cells compared to controls was observed on the 3rd, 7th, and 14th days following irradiation, but not on the 1st day. Consistent with these results, the proliferating cell nuclear antigen labeling index of irradiated cells decreased significantly on the 3rd, 7th, and 14th days following irradiation, but not on the 1st day. By comparison, the regrowth area on day 14 only showed no difference compared to the control group. The apoptotic index increased on the 7th and 14th day after irradiation, but at a lower rate than the observed decrease in the level of proliferating cell nuclear antigen. We speculate that the major mechanism of single high-dose radiation effect is inhibition of DNA synthesis.

Potent preventive action of curcumin on radiation-induced initiation of mammary tumorigenesis in rats.

This investigation evaluated the preventive effect of curcumin on radiation-induced tumor initiation in rat mammary glands. Fifty-four female rats were mated and then divided into two groups at day 11 of pregnancy. As the control group, 27 rats were fed a basal diet during the experimental period. As the experimental group, 27 rats were fed a diet containing 1% curcumin between day 11 of pregnancy and parturition (day 23 of pregnancy). All rats of both groups received whole body irradiation with 1.5 Gy gamma-rays from a (60)Co source at day 20 of pregnancy and were then implanted with a diethylstilbestrol pellet 1 month after weaning. A high incidence (70.3%) of mammary tumorigenesis was observed in the control group. The tumor incidence (18.5%) was significantly reduced in the rats fed curcumin during the initiation stage. The appearance of the first palpable tumor was delayed by 6 months in the curcumin-fed group and the average latent period until the appearance of mammary tumors was 2.5 months longer in the curcumin-fed group than in the control group. By histological examination, the proportion of adenocarcinoma (16.7%) in total tumors in the curcumin-fed rats was found to be decreased to half that (32.1%) in the control group. Compared with the control rats, the body weight of rats in the experimental group was decreased slightly by administration of the curcumin diet from day 11 of pregnancy, in spite of a similar intake of diet, but had recovered to the level of the control by the end of the experiment. At the time of irradiation, curcumin did not have any effect on organ weight or on the development and differentiation of mammary glands of pregnant rats. In addition, the serum concentrations of fatty acids, thiobarbituric acid-reactive substances and ovarian and pituitary hormones, except LH, remained at the control level. Also, no change in litter size and body weight of pups born from curcumin-fed rats indicated no toxicity of curcumin. These results suggest that curcumin does not have any side-effects and is an effective agent for chemoprevention acting at the radiation-induced initiation stage of mammary tumorigenesis.

Histologic evaluation of rat mammary tumor necrosis by interstitial Nd:YAG laser hyperthermia.

The extent of coagulative necrosis caused by interstitial laser hyperthermia was measured for different quantities of laser energy in a rat mammary tumor model. Continuous wave Nd:YAG laser at a power level of 5 W was focused onto a 600 mu diameter bare tip quartz fiber and placed inside a 19-gauge needle, which allowed the para-axial flow of normal saline at 1 cc/min. A microthermocouple soldered to the outside of the probe continuously provided the interstitial temperature. After the probe was inserted into the tumor, it was withdrawn as laser energy was administered at a rate sufficient to maintain the temperature within 42-45 degrees C. Tumors were excised after 48 hours, fixed in formalin, cut in 3 mm slices, and the coagulated surfaces measured microscopically. Laser fiber transmission loss was 1% per 1,000 J of laser energy and the average time required to coagulate 1 cc of tumor was 2 minutes. There was a statistically significant correlation between the volume of tumor necrosis and the level of laser irradiation (r = 0.71, P less than 0.001). It is concluded that the described technique is an efficient method of tumor coagulation by interstitial laser hyperthermia and proportionally larger volumes of necrosis are created with greater amounts of laser energy.