Effects of Combined Exposure to Carbon Ions and Protons on the Pool of MCF-7 Breast Cancer Stem Cells In Vitro.

We studied the effects of single and combined action of protons and carbon ions 12C6+ on the pool of MCF-7 human breast cancer stem cells. Single irradiation with a beam of protons or carbon ions had no significant effects on the relative number of cancer stem cells (CSC). The effects of combined irradiation in a total equieffective dose of 4 Gy depended on the sequence of exposure to ionizing radiations: the relative number of CSC did not change after irradiation with carbon ions and then with protons, but increased in the case of the reverse sequence. The most favorable result, i.e. a decrease in the CSC pool, was observed in the case of sequential irradiation with carbon ions and protons and their equal contribution to total equieffective dose. In this case, the absolute number of CSC decreased by on average 2.1 times in comparison with the control (p<0.05). The revealed regularities are of interest for the further development of new methods of radiation therapy.

Effects of Combined Action of Neutron and Proton Radiation on the Pool of Breast Cancer Stem Cells and Expression of Stemness Genes In Vitro.

Radiation response of cancer stem cells was studied in two breast cancer cell lines: luminal A (MCF-7) and triple negative (MDA-MB-231) subtypes. The relative number of CD44+CD24-/low cancer stem cells of the MCF-7 line increased by 1.7 times under the influence of γ-irradiation at a dose of 4 Gy (p=0.047 in comparison with the control). However, no significant changes were found in the relative number of cancer stem cells and the expression level of OCT4, NANOG, and SOX2 genes after neutron, proton beam irradiation or their combined action at a total equieffective dose of 4 Gy in comparison with the control in both cell lines. The absolute number of cancer stem cells decreased under the influence of neutron or proton radiation in comparison with the control (p<0.05 for both cell lines). At the same time, the effects of sequential exposures to neutron and proton radiation on the size of the cancer stem cell pool depended on the molecular subtype of cancer cells. Additive interaction was observed for MCF-7 line and antagonistic one for MDA-MB-231 line (coefficients of synergism were 0.96 and 0.45, respectively).

Correlation of Radiation Response of Cervical Cancer Stem Cells with Their Initial Number before Treatment and Molecular Genetic Features of Papillomavirus Infection.

The proportion of CD44+CD24low cancer stem cells (CSC) was determined in cervical scrapings of 41 patients with squamous cell carcinoma of the uterine cervix before treatment and after irradiation in a total focal dose of 10 Gy. The relationship of quantitative changes in the CSC population with such parameters of papillomavirus infection as genotype, viral load, and physical status of HPV DNA (the absence or presence of HPV DNA integration into the cell genome and the degree of integration) was studied. Single- and multi-factor analysis revealed 2 independent indicators affecting the radiation response of CSC: initial number of these cells before treatment and physical status of HPV DNA. The increase in the CSC proportion after radiation exposure was observed 4.5-fold more often in patients with an initially low proportion of CSC (<3%) than that in other patients (p=0.001). The CSC proportion increased by on average 3% after irradiation in patients with complete integration of HPV 16/18 DNA and decreased by 3.8 % in patients with partial integration or no integration (p=0.03).

Quantitative Changes in the Population of Cancer Stem Cells after Radiation Exposure in a Dose of 10 Gy as a Prognostic Marker of Immediate Results of the Treatment of Squamous Cell Cervical Cancer.

Prognostic significance of the proportion of cancer stem cells in cervical scrapings from 38 patients with uterine cervical cancer before treatment and after irradiation in a total dose of 10 Gy was assessed for immediate results of radio- and combined chemoradiotherapy evaluated by the degree of tumor regression in 3-6 months after the treatment. Cancer stem cells were detected as cells with CD44+CD24low immunophenotype by flow cytometry. The proportion of cancer stem cells in patients with the complete tumor regression decreased by on average 2.2±1.1% after irradiation, while in patients with partial regression this indicator increased by on average 3.3±2.3% (p=0.03). Multiple regression analysis revealed two independent indicators affecting tumor regression: the stage of the disease (which is quite expected) and change in the proportion of cancer stem cells after the first irradiation sessions (R=0.60, p<0.002 for the model in the whole). The proportion of cancer stem cells before the treatment did not have prognostic significance.

[Accumulation of doxorubicin conjugates with dendritic polymer and vector protein in normal and tumor cells in vitro].

Accumulation of doxorubicin (Dox), its conjugates with the second generation dendritic polymer (G2-Dox) and vector pro- tein (recombinant third domain of alpha-fetoprotein - 3D-G2- Dox) in normal and tumor cells was studied in vitro within the framework of the development of selective transport system of anticancer drugs to the target cells. The objects of the study were cells of peripheral blood mononuclear fraction of healthy donors and cells of breast adenocarcinoma lines MCF-7 and MCF-7/MDR1, differing in chemosensitivity. G2-Dox and 3D-G2-Dox accumulated in tumor cells of the both lines better than free Dox (p<0,05). However removal of these drugs out of cells MCF-7 and MCF-7/MDR1 was significantly different: in the latter case all free Dox was excluded from the cells for 24 hours while Dox, accumulated in composition with dendrimers, still remained in the cells. It was important that 3D-G2-Dox (unlike the G2-Dox) accumulated in normal cells worse than free Dox (p<0.01). Thus, the results indicate that the use of 3D-G2-Dox is the most promising because it accumulates in tumor cells better and in normal cells worse than free Dox. Furthermore it can be assumed that the use of 3D-G2-Dox would be especially useful in cases of multi-drug resistance associated with the high expression of P-glycoprotein.

High Level of Radiation-Induced Heat Shock Protein with a Molecular Weight of 27 and 70 kDa is the Hallmark of Radioresistant SP Cells of MCF-7 Breast Cancer Culture.

As previously indicated, side population cells (side population, SP) of breast cancer line MCF-7 have greater resistance to the action of low-LET radiation compared to other tumor cells (non SP, NSP). One can assume that one possible reason for the high radioresistance of this fraction of tumor cells is the increased expression of different heat shock proteins (HSP) before and/or after radiation exposure. To verify this hypothesis, we investigated the expression of HSP27 and HSP70 in these populations of cells-before and after irradiation at a dose of 5.0 Gy. The study was performed using scanning microscopy for NSP and SP cells after sorting and immunocytochemical staining. A substantial increase of HSP27 and HSP70 in SP cells was found after irra- diation as'compared with the control. In NSP cells the HSP27 level increased in response to radiation exposure, but to a lesser extent than in SP cells, while the content of HSP70 did not change after irradiation. The results confirm the assumption about HSP27 and HSP70 participation in the formation of SP cell radioresistance by the example of MCF-7 line.

Changes in the Relative Number of SP Cells of Melanoma Line B16 after Radiation Exposure in vivo.

The quantitative regularities of changes of cancer stem cell (CSC) population were explored after local γ-irradiation of experimental tumors (murine melanoma line B16). CSCs were detected by the ability of these cells to exclude Hoechst 33342 fluorescent dye and to form a so-called side population (SP) under flow cytometry study. In the control group of unexposed mice a positive correlation was found between the proportion of CSCs (SP) and tumorweight at the initial stage of growth (R = 0.77, p = 0.009). In the advanced stages of tumor growth similar relationship was not revealed. Statistically significant increase in the proportion of CSCs (SP) occurred 2-5 days after exposure of tumor to a dose of 10 Gy as compared to control; this index returned to the control level 8 days after irradiation. On the second day after exposure to radiation a linear correlation between the percentage of CSCs and a radiation dose in the range of 2-10 Gy was established (R = 0.98, p = 0.003), confirming a higher radioresistance of this population as compared to other cells not only in vitro (as it was previously shown by us and other authors), but also in vivo. These results suggest the possibility of application of this model system to assess the CSC sensitivity to various antitumor agents in vivo, including preclinical trials, and clarify the details of the practical application of this method.

[The Combined Effects of Ionizing Radiation and Dendritic Polymers Loaded with Doxorubicin on the MCF-7 Breast Cancer Cell Line].

The dendritic polymers (dendrimers) are perspective nanocontainers for transportation of anticancer drugs into cells and a controlled release of the delivered substances. However, the combined effect of ionizing radiation and dendrimers loaded with anticancer drugs has been poorly studied and is the aim of this research. We used poliamidoamin (PAMAM) dendrimers of the second generation (G2) covalently conjugated with doxorubicin (Dox) via an acid labile linker, cis-aconitic anhydride. We compared the intracellular accumulation of Dox and growth rate of the MCF-7 cell culture under the single and combined action of ionizing radiation at a dose of 4 Gy, free Dox and G2-Dox. It was found that within 2 hours free Dox accumulated in cancer cells better than Dox connected with G2 dendrimers (p < 0.05 in the concentration range of 1-5 µmol/l). The intracellular accumulation of Dox was higher by 1.7 times for the free Dox than that connected with dendrimers (for concentration 0.5 µmol/l p = 0.02) after 26 hours of incubation. Like the intracellular accumulation of Dox, inhibition of the cell culture growth was more pronounced when using free Dox than G2-Dox in the case of both a single and combined action of these drugs. Subadditivity effects of the combined action of both drugs and ionizing radiation are shown in terms of reducing the number of tumor cells 24 hours after irradiation. The results indicate the need for further development of selective delivery systems for Doxin tumor cells, providing a more intense accumulation of anticancer drug in target cells.

[Increase in the number of cancer stem cells after exposure to low-LET radiation].

Radioresistance of cancer stem cells (CSCs) is regarded as one of the possible causes of cancer recurrence after radiotherapy. Since the regularities and mechanisms of radiation effects on this population of cells have not been sufficiently studied, the aim of this work is to elucidate the changes in the CSC number after γ-irradiation in stable cultures of tumor cells in vitro and tumor tissue in vivo (in the course of radiation therapy of patients with cancers of the upper respiratory tract). CSCs were identified in the cell lines B16, MCF-7, HeLa by the ability to exclude the fluorescent dye Hoechst 33342 (SP method) 48-72 h after irradiation at the doses of 1-20 Gy and in biopsy material by immunophenotype CD44+CD24(-/low) before and 24 h after irradiation at the total dose of 10 Gy. The essential differences in the response of CSCs and other cancer cells were found after exposure to low-LET radiation. The absolute number of CSCs increased after a single exposure at the doses ranging from 1 to 5-10 Gy in different cell cultures, but a further dose increase maintained the current number of CSCs or decreased it. At the same time, the number of non CSCs significantly decreased with increasing doses of radiation exposure, as expected. Fractionated irradiation in vivo at a total dose of 10 Gy increased the relative amount of CSCs in most patients. The registered changes are an integral indicator of cell death, cell division delay immediately after irradiation, proliferation at a later time, possible dedifferentiation of non CSCs, etc. The exact contribution of each of them to the radiation-induced increase of the CSCs number is of considerable interest and requires further research.

[Influence of systemic photodynamic therapy on circulating tumor cells in peripheral blood of cancer patients].

The aim of this investigation is to study the number of circulating tumor cells (CTCs) in the blood of cancer patients after systemic photodynamic therapy (PDT) at different times and to assess apoptosis of these cells. The study group consisted of 19 patients with malignant tumors of epithelial origin at various stages (II-IV). CTC identification was performed with flow cytometry by immunophenotype Ep-CAM (CD326)+ CD45-. CTC apoptosis was identified by criteria of plasma membrane integrity and phosphatidylserine translocation on the outer surface of the membrane. Negative correlation between the CTC frequency and apoptotic death rate of these cells was found in patients before the treatment (R = -0.51, p = 0.03). CTC frequency gradually reduced during the first three days after PDT, and then it was maintained at the same level until the end of the follow-up (7 days). At the individual level, the effect of PDT depended on the frequency of CTCs before the treatment: the decrease in these cell frequency occurred significantly more often in the patients with an initially high frequency of CTCs than in other patients (p = 0.05). With the decrease in the CTC frequency, apoptotic death increased within 6 hours after the treatment and remained at the same level until the end of the follow-up period. The results demonstrate the efficacy of systemic PDT for elimination of tumor cells circulating in the peripheral blood of cancer patients with different localization of primary tumor and stage of disease.

[Radioresistance mechanisms of side population cells in mouse melanoma cell line B16].

As it was shown by us earlier, side population (SP) cells are more resistant to the low-LET radiation than the other part of mouse melanoma B16 cells (Matchuk et al., 2012). The aim of our research was finding some mechanisms of radioresistance, therefore we analyzed SP and nonSP cell cycle distribution, spontaneous and radiation induced DNA double-strand breaks (number of γH2AX foci) and intracellular NO concentration. The results indicate that SP cells have significantly less DNA double-strand breaks after irradiation at dose of 3 Gy than nonSP cells (24.4 vs 40.3, accordingly, P < 0.05 Mann-Whitney Ucriterion). SP cells are more quiescent compared to nonSP G1/G0 fraction is 85 vs 39%, accordingly, P < 0.01 Mann-Whitney U criterion). Most nonSP cells reside in S, G2/M phases (61%), believed to be rather radiosensitive. Thus, the difference of SP and nonSP cells radiosensitivity can be partly explained by peculiarities of cell cycle distribution. NO concentration is 1.5 times higher in SP than nonSP cells (P < 0.05 Mann-Whitney U criterion); since it is known that NO inhibits apoptosis, being one of the mechanisms of genetic stability maintenance, greater number of spontaneous DNA double-strand breaks in SP cells is unsurprising (P < 0.05 Mann-Whitney U criterion). The above-listed results explain considerably the higher resistance of SP cells to the action of low-LET radiation in comparison with other melanoma B16 cells. Further study of this question can become the basis for development of tools to target SP cells and, ultimately, more effective cancer treatment.

[Sensitivity of melanoma B16 side population to low- and high-LET radiation].

Cancer stem cells (CSC) found in multiple tumor types and cancer cell lines were shown to be more resistant to low-LET radiation in comparison to other cancer cells. Therefore, CSC are supposed to determine the long-term effect of cancer therapy. Research into the CSC sensitivity to high-LET radiation is of great interest because of the advances in hadron therapy. The aim of this investigation is to compare CSC and other cancer cell sensitivity to the low- (60Co gamma-rays) and high-LET (neutron) radiation. To identify CSC, we used the low cytometry-based side population (SP) technique based on the CSC capacity to produce the efflux of the vital dye Hoechst 33342. SP and non SP cells were sorted and exposed to gamma and neutron radiation at doses of 1-10 Gy and 0.1-4.7 Gy, correspondingly. We applied the colony-formation test to examine the SP and non SP survival rate after irradiation. It was shown that the sensitivity of SP to gamma-irradiation was lower than that of other cells: D0 average values (+/- SE) made up 2.3 +/- 0.3 Gy and 1.4 +/- 0.2 Gy, correspondingly (p = 0.047). The survival rate of SP and non SP did not differ after neutron irradiation. The values of relative biological effectiveness of neutron radiation relative to gamma-radiation at the D10 level were 2.6 for SP and 2.1 for other cells. The obtained results justify for the first time a high efficiency of application of neutrons in radiotherapy from the point of view of CSC elimination.