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.

Targeting DNA double strand break repair with hyperthermia and DNA-PKcs inhibition to enhance the effect of radiation treatment.

Radiotherapy is based on the induction of lethal DNA damage, primarily DNA double-strand breaks (DSB). Efficient DSB repair via Non-Homologous End Joining or Homologous Recombination can therefore undermine the efficacy of radiotherapy. By suppressing DNA-DSB repair with hyperthermia (HT) and DNA-PKcs inhibitor NU7441 (DNA-PKcsi), we aim to enhance the effect of radiation.The sensitizing effect of HT for 1 hour at 42°C and DNA-PKcsi [1 µM] to radiation treatment was investigated in cervical and breast cancer cells, primary breast cancer sphere cells (BCSCs) enriched for cancer stem cells, and in an in vivo human tumor model. A significant radio-enhancement effect was observed for all cell types when DNA-PKcsi and HT were applied separately, and when both were combined, HT and DNA-PKcsi enhanced radio-sensitivity to an even greater extent. Strikingly, combined treatment resulted in significantly lower survival rates, 2 to 2.5 fold increase in apoptosis, more residual DNA-DSB 6 h post treatment and a G2-phase arrest. In addition, tumor growth analysis in vivo showed significant reduction in tumor growth and elevated caspase-3 activity when radiation was combined with HT and DNA-PKcsi compared to radiation alone. Importantly, no toxic side effects of HT or DNA-PKcsi were found.In conclusion, inhibiting DNA-DSB repair using HT and DNA-PKcsi before radiotherapy leads to enhanced cytotoxicity in cancer cells. This effect was even noticed in the more radio-resistant BCSCs, which are clearly sensitized by combined treatment. Therefore, the addition of HT and DNA-PKcsi to conventional radiotherapy is promising and might contribute to more efficient tumor control and patient outcome.

Dynamics of chromosomal aberrations, induction of apoptosis, BRCA2 degradation and sensitization to radiation by hyperthermia.

Hyperthermia can transiently degrade BRCA2 and thereby inhibit the homologous recombination pathway. Induced DNA-double strand breaks (DSB) then have to be repaired via the error prone non-homologous end-joining pathway. In the present study, to investigate the role of hyperthermia in genotoxicity and radiosensitization, the induction of chromosomal aberrations was examined by premature chromosome condensation and fluorescence in situ hybridisation (PCC-FISH), and cell survival was determined by clonogenic assay shortly (0-1 h) and 24 h following exposure to hyperthermia in combination with ionizing radiation. Prior to exposure to 4 Gy γ-irradiation, confluent cultures of SW­1573 (human lung carcinoma) and RKO (human colorectal carcinoma) cells were exposed to mild hyperthermia (1 h, 41˚C). At 1 h, the frequency of chromosomal translocations was higher following combined exposure than following exposure to irradiation alone. At 24 h, the number of translocations following combined exposure was lower than following exposure to irradiation only, and was also lower than at 1 h following combined exposure. These dynamics in translocation frequency can be explained by the hyperthermia-induced transient reduction of BRCA2 observed in both cell lines. In both cell lines exposed to radiation only, potentially lethal damage repair (PLDR) correlated with a decreased number of chromosomal fragments at 24 h compared to 1 h. With combined exposure, PLDR did not correlate with a decrease in fragments, as in the RKO cells at 24 h following combined exposure, the frequency of fragments remained at the level found after 1 h of exposure and was also significantly higher than that found following exposure to radiation alone. This was not observed in the SW­1573 cells. Cell survival experiments demonstrated that exposure to hyperthermia radiosensitized the RKO cells, but not the SW­1573 cells. This radiosensitization was at least partly due to the induction of apoptosis, which was only observed in the RKO cells and which may have been induced by BRCA2 degradation or different types of chromosomal aberrations. An important observation of this study is that the genotoxic effect of hyperthermia shortly after combined epxosure (to hyperthermia and radiation) is not observed at 24 h after treatment.

Hyperthermia Selectively Targets Human Papillomavirus in Cervical Tumors via p53-Dependent Apoptosis.

Human papillomavirus (HPV) is associated with cervical cancer, the third most common cancer in women. The high-risk HPV types 16 and 18 are found in over 70% of cervical cancers and produce the oncoprotein, early protein 6 (E6), which binds to p53 and mediates its ubiquitination and degradation. Targeting E6 has been shown to be a promising treatment option to eliminate HPV-positive tumor cells. In addition, combined hyperthermia with radiation is a very effective treatment strategy for cervical cancer. In this study, we examined the effect of hyperthermia on HPV-positive cells using cervical cancer cell lines infected with HPV 16 and 18, in vivo tumor models, and ex vivo-treated patient biopsies. Strikingly, we demonstrate that a clinically relevant hyperthermia temperature of 42 °C for 1 hour resulted in E6 degradation, thereby preventing the formation of the E6-p53 complex and enabling p53-dependent apoptosis and G2-phase arrest. Moreover, hyperthermia combined with p53 depletion restored both the cell-cycle distribution and apoptosis to control levels. Collectively, our findings provide new insights into the treatment of HPV-positive cervical cancer and suggest that hyperthermia therapy could improve patient outcomes.

Chromosome fragments have the potential to predict hyperthermia-induced radio-sensitization in two different human tumor cell lines.

Cellular radiosensitivity, assessed by loss of clonogenicity, has been shown to correlate with the number of radiation-induced chromosomal aberrations. Also an increased radiosensitivity by hyperthermia has been shown to correlate with an increase in chromosomal aberrations. Therefore, determination of the number of chromosomal aberrations might be used as an assay to predict the radiosensitivity of tumors pre-treated with hyperthermia at clinically relevant temperatures. The use of premature chromosome condensation combined with fluorescent in situ hybridisation (PCC-FISH) has been shown to be clinically applicable. Therefore, the use of chromosomal aberrations as determined with PCC-FISH for the prediction of hyperthermia-induced radio-sensitization in human tumor cells was investigated. Confluent cultures of SW-1573 (human lung carcinoma) and RKO (human colorectal carcinoma) cells were treated with 1 h 41 degrees C or 43 degrees C hyperthermia prior to gamma-irradiation. Clonogenic cell survival and induction of chromosomal aberrations (unrejoined chromosomal fragments and translocations), by PCC-FISH, were studied at 24 h after treatment. Pre-treatment with hyperthermia at 41 degrees C for 1 h enhanced the radiosensitivity of RKO cells but not of SW-1573 cells. Increasing the temperature to 43 degrees C for 1 h enhanced the radiosensitivity of SW-1573 cells. When radio-sensitization was observed, a significant increase in the number of unrejoined chromosomal fragments was found but the frequency of translocations was not increased. Hyperthermia-induced radio-sensitization is correlated with an increase in unrejoined chromosomal fragments. This suggests that determination of the number of chromosomal fragments after hyperthermia and radiation treatment might be used for the prediction of combined treatment response in cancer patients.

Effect of 41 degrees C and 43 degrees C on cisplatin radiosensitization in two human carcinoma cell lines with different sensitivities for cisplatin.

The effect of trimodality treatment consisting of hyperthermia, cisplatin and radiation was investigated in two cell lines with different sensitivities to cisplatin. Hyperthermia treatment was performed for 1 h at 41 degrees C and 43 degrees C in order to compare the effects of the two temperatures. Clonogenic assays were performed with cisplatin-sensitive SiHa human cervical carcinoma and cisplatin-resistant SW-1573 human lung carcinoma cell lines. Cells were treated with various combinations of hyperthermia, cisplatin and radiation. Radiation was performed after 1 h of simultaneous hyperthermia and cisplatin treatment. Cisplatin exposure was for 1 h or continuous without refreshment of the cisplatin-containing medium. SiHa cells were more sensitive to cisplatin than SW-1573 cells. Hyperthermia at 41 degrees C decreased survival in SW-1573 cells but was not cytotoxic in SiHa cells. Hyperthermia at 43 degrees C decreased survival dramatically in both cell lines with SiHa being the most sensitive. The addition of hyperthermia at 41 degrees C and 43 degrees C to cisplatin treatment led to enhanced cell kill in both cell lines compared with cisplatin alone. Radiosensitization was observed after continuous but not after 1 h of cisplatin treatment. Hyperthermia at 43 degrees C increased radiosensitivity whereas hyperthermia at 41 degrees C did not. A combination of 41 degrees C hyperthermia with continuous cisplatin treatment had an additive effect on SW-1573 cells but enhanced cisplatin radiosensitivity of SiHa cells. In SW-1573 cells trimodality treatment using 43 degrees C hyperthermia enhances cisplatin radiosensitivity. We conclude that hyperthermia at 43 degrees C enhances cisplatin-induced radiosensitization in both cisplatin-sensitive and -resistant cell lines. Hyperthermia at 41 degrees C was also able to increase cisplatin-induced radiosensitivity but only in the cisplatin-sensitive SiHa cell line.