Effect of proton therapy and chemoradiotherapy on biochemistry of radioresistant prostate cancer cells studied by Raman microspectroscopy.

Euchromatic histone lysine methyltransferase 2 facilitates radioresistance in prostate cancer by repressing endoplasmic reticulum protein 29 transcription.

Comparison of biochemical changes induced in radioresistant prostate cancer cells by X-rays, radiosensitizing drugs, and a combined therapy using Raman microspectroscopy

Objective: To investigate the role of hypoxia-inducible factor-1α (HIF-1α) and β-catenin in radioresistance of prostate cancer (PCa) cells. Method: Two PCa cell lines, LNCaP and C4-2B, were grouped as: negative control (no treatment), HIF-1α overexpression group (transfected with HIF-1α plasmids), and β-catenin silencing group (transfected with HIF-1α plasmids and β-catenin-shRNA). Cell proliferation, cycle, invasion, and radiosensitivity were measured under normal or hypoxic condition. Radiosensitivity was tested in two mice PCa models (the LNCaP orthotopic BALB/c-nu mice model and the C4-2B subcutaneous SCID mice model). Results: In both LNCaP and C4-2B cells, HIF-1α transfection led to an enhanced β-catenin nuclear translocation, while β-catenin silencing inhibited the β-catenin nuclear translocation. Enhanced β-catenin nuclear translocation caused by HIF-1α overexpression resulted in enhanced cell proliferation and invasion, altered cell cycle distribution, reduced apoptosis, and improved non-homologous-end-joining (NHEJ) repair under irradiation condition. In vivo imaging of orthotopic models showed that HIF-1α overexpression LNCaP cells produced tumors with 3-fold volume (P=0.003 1) and 2-fold wet weight (P=0.039 4) than those by negative control cells at day 21, and β-catenin silencing cells aberrantly reduced both tumor volume (P=0.000 3) and wet weight (P=0.017 5) than HIF-1α overexpression cells. In addition, C4-2B subcutaneous models showed similar tumor promotion effects induced by HIF-1α overexpression (tumor volume: P=0.000 1 and wet weight: P=0.047 3) and suppressive effects by β-catenin silencing (tumor volume: P<0.000 1 and wet weight: P=0.022 1) as LNCaP orthotopic xenograft with regard to tumor volume and wet weight. Conclusions: HIF-1α overexpression enhanced β-catenin nuclear translocation, which led to the activation of the β-catenin/NHEJ signaling pathway and increased cell proliferation, invasion, and DNA repair. These results suggest that HIF-1α overexpression led to radioresistance of PCa cells.

Radiotherapy is an important treatment modality for patients with esophageal cancer; however, the response to radiation varies among different tumor subpopulations due to tumor heterogeneity. Cancer cells that survive radiotherapy (i.e., radioresistant) may proliferate, ultimately resulting in cancer relapse. However, the interaction between radiosensitive and radioresistant cancer cells remains to be elucidated. In this study, we found that the mutual communication between radiosensitive and radioresistant esophageal cancer cells modulated their radiosensitivity. Radiosensitive cells secreted more exosomal let-7a and less interleukin-6 (IL-6) than radioresistant cells. Exosomal let-7a secreted by radiosensitive cells increased the radiosensitivity of radioresistant cells, whereas IL-6 secreted by radioresistant cells decreased the radiosensitivity of radiosensitive cells. Although the serum levels of let-7a and IL-6 before radiotherapy did not vary significantly between patients with radioresistant and radiosensitive diseases, radiotherapy induced a more pronounced decrease in serum let-7a levels and a greater increase in serum IL-6 levels in patients with radioresistant cancer compared to those with radiosensitive cancer. The percentage decrease in serum let-7a and the percentage increase in serum IL-6 levels at the early stage of radiotherapy were inversely associated with tumor regression after radiotherapy. Our findings suggest that early changes in serum let-7a and IL-6 levels may be used as a biomarker to predict the response to radiotherapy in patients with esophageal cancer and provide new insights into subsequent treatments.

Resistance to chemo- or radiotherapy is the main obstacle to consistent treatment outcomes in oncology patients. A deeper understanding of the mechanisms driving the development of resistance is required. This review focuses on secretory factors derived from chemo- and radioresistant cancer cells, cancer-associated fibroblasts (CAFs), mesenchymal stem cells (MSCs), and cancer stem cells (CSCs) that mediate the development of resistance in unexposed cells. The first line of evidence considers the experiments with conditioned media (CM) from chemo- and radioresistant cells, CAFs, MSCs, and CSCs that elevate resistance upon the ionizing radiation or anti-cancer drug exposure of previously untreated cells. The composition of CM revealed factors such as circular RNAs; interleukins; plasminogen activator inhibitor; and oncosome-shuttled lncRNAs, mRNAs, and miRNAs that aid in cellular communication and transmit signals inducing the chemo- and radioresistance of sensitive cancer cells. Data, demonstrating that radioresistant cancer cells become resistant to anti-neoplastic drug exposure and vice versa, are also discussed. The mechanisms driving the development of cross-resistance between chemotherapy and radiotherapy are highlighted. The secretion of resistance-mediating factors to intercellular fluid and blood brings attention to its diagnostic potential. Highly stable serum miRNA candidates were proposed by several studies as prognostic markers of radioresistance; however, clinical studies are needed to validate their utility. The ability to predict a treatment response with the help of the miRNA resistance status database will help with the selection of an effective therapeutic strategy. The possibility of miRNA-based therapy is currently being investigated with ongoing clinical studies, and such approaches can be used to alleviate resistance in oncology patients.

High linear energy transfer (LET) radiation or heavy ion such as carbon ion radiation is used as a method for advanced radiotherapy in the treatment of cancer. It has many advantages over the conventional photon based radiotherapy using Co-60 gamma or high energy X-rays from a Linear Accelerator. However, charged particle therapy is very costly. One way to reduce the cost as well as irradiation effects on normal cells is to reduce the dose of radiation by enhancing the radiation sensitivity through the use of a radiomodulator. PNKP (polynucleotide kinase/phosphatase) is an enzyme which plays important role in the non-homologous end joining (NHEJ) DNA repair pathway. It is expected that inhibition of PNKP activity may enhance the efficacy of the charged particle irradiation in the radioresistant prostate cancer cell line PC-3. To test this hypothesis, we investigated cellular radiosensitivity by clonogenic cell survival assay in PC-3 cells.12Carbon ion beam of62 MeVenergy (equivalent 5.16 MeV/nucleon) and with an entrance LET of 287 kev/μm was used for the present study. Apoptotic parameters such as nuclear fragmentation and caspase-3 activity were measured by DAPI staining, nuclear ladder assay and colorimetric caspase-3method. Cell cycle arrest was determined by FACS analysis. Cell death was enhanced when carbon ion irradiation is combined with PNKPi (PNKP inhibitor) to treat cells as compared to that seen for PNKPi untreated cells. A low concentration (10μM) of PNKPi effectively radiosensitized the PC-3 cells in terms of reduction of dose in achieving the same survival fraction. PC-3 cells underwent significant apoptosis and cell cycle arrest too was enhanced at G2/M phase when carbon ion irradiation was combined with PNKPi treatment. Our findings suggest that combined treatment of carbon ion irradiation and PNKP inhibition could enhance cellular radiosensitivity in a radioresistant prostate cancer cell line PC-3. The synergistic effect of PNKPi and carbon ion irradiation could be used as a promising method for carbon-ion therapy in radioresistant cells.

Resistance to radiotherapy is one of the main obstacles to improving cancer prognoses. To effectively destroy cancer cells, novel radiation sensitizers are needed. Recently, several natural products have been shown to exhibit promising tumor-killing properties. However, little is known about the specific mechanisms of these natural compounds on cancer treatment. In this study, after screening a high-throughput natural product library, we identified tanshinone I (Tan I) as a potential radiation sensitizer in lung cancer cells. Lung cancer radioresistant cell lines, H358-IR and H157-IR, were first established to confirm the radioresistant phenotypes. After that, a natural product library was used to screen the potential radiation sensitizer. We further examined the inhibition functions of Tan I on radioresistant cancer cells via a series of experiments. Tan I significantly inhibited cell proliferation and clone formation, consequently enhancing radiosensitivity in radioresistant lung cancer cells, H358-IR and H157-IR. Stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative proteomics indicated that Tan I downregulates expression of pro-oncogenic protein phosphoribosyl pyrophosphate aminotransferase (PPAT) in both H358-IR and H157-IR cells. Further analysis of molecular docking showed that Tan I is well-docked into the active pocket of the structure of PPAT, serving as a potential PPAT inhibitor. Taken together, these findings suggest that inhibition of the tumor promoter PPAT by Tan I exerts marked inhibitory effects on radioresistant lung cancer cells, improving radiation efficacy.

Background. Radiation therapy is one of the leading treatments for early and late stage prostate cancer. Radiation therapy is one of the leading treatments for early and late stage prostate cancer. The significant frequency of prostate cancer progression after radiation therapy makes it relevant to study the molecular mechanisms of the development of radioresistance, to identify prognostic markers of its development.Objective: identification and analysis of the mechanism of action of microRNAs regulating radioresistance of prostate cancer cells on the model of the androgen-independent DU145 cell line.Materials and methods. We used human prostate adenocarcinoma cell lines: DU145-hormone-independent prostate cancer cell line and DU145-RR - its radioresistant variant. Differential microRNA expression was measured in cultured DU145 and DU145-RR cells 1, 8 days after a single gamma irradiation at a dose of 4 Gy. To analyze the differential expression of microRNAs in the initial and radioresistant variants of DU145 cells, the HiSeq 2000 platform (Illumina Inc., USA) was used. The miRBase v.21 database was used to identify microRNAs. The miRTarbase 7.0 and KEGG PATHWAY databases were used for bioinformatic analysisResults. The results of the study showed that the aberrant expression of miR-101-3p, -148a-3p, -21-3p, -532-5p, -92a-3p in DU145-RR cells upregulated compared to that in DU145 cells, and miR-125b-5p, -23a-3p, -424-3p - downregulated. It has been shown that the role of these microRNAs is associated with the provision of functional interaction between DNA methyltransferases, the transcriptional regulator of the proto-oncogenic protein Myc, and PTEN phosphatase in the regulation of the activity of MAPK and PI3K protein kinase signaling cascades. Constitutive activation of these cascades leads to an increase in cell survival, migration, proliferation, and growth.Conclusion. A wide range of target genes and a significant change in the expression profiles of microRNAs in various conditions, including the transition of malignant cells to a radioresistant status, makes microRNAs promising prognostic markers of radioresistance in prostate cancer.

Breast cancer is the most common type of cancer with high incidence in women. Currently, identifying new therapies that selectively inhibit tumor growth without damaging normal tissue are a major challenge of cancer research. One of the features of cancer cells is that they do not consume more oxygen even under normal oxygen circumstances but prefer to aerobic glycolysis through the enhanced catabolism of glucose and glutamine. In this study, we investigate the mechanisms of the radioresistance in breast cancer cells. Human breast cancer cells MDA-MB-231 and MCF-7 were treated with radiation alone, Glut1 inhibitor alone or the combination of both to evaluate cell glucose metabolism and apoptosis. By the establishment of radioresistant cell line, we investigate the mechanisms of the combined treatments of radiation with Glut1 inhibitor in the radioresistant cells. The glucose metabolism and the expression of Glut1 are significantly stimulated by radiotherapy. We report the radioresistant breast cancer cells exhibit upregulated Glut1 expression and glucose metabolism. In addition, we observed overexpression of Glut1 renders breast cancer cells resistant to radiation and knocking down of Glut1 sensitizes breast cancer cells to radiation. We treated breast cancer cells with radiation and WZB117 which inhibits Glut1 expression and glucose metabolism and found the combination of WZB117 and radiation exhibits synergistically inhibitory effects on breast cancer cells. Finally, we demonstrate the inhibition of Glut1 re-sensitizes the radioresistant cancer cells to radiation. This study reveals the roles of Glut1 in the radiosensitivity of human breast cancer. It will provide new mechanisms and strategies for the sensitization of cancer cells to radiotherapy through regulation of glucose metabolism.

Radiation is a therapeutic strategy for the treatment of cancer, and is also used for the treatment of hepatocellular carcinoma. MicroRNAs (miRs) are endogenous, non‑coding single‑stranded RNA molecules, which regulate gene expression at the post‑transcriptional level. In the present study, the roles of miR‑34a‑mediated glycolysis in radiation sensitivity were investigated. By establishing a radioresistant liver cancer cell line, the present study compared the expression level of miR‑34a from radiosensitive and radioresistant cells using the reverse transcription‑quantitative polymerase chain reaction. The glucose uptake and lactate production were also compared between the two types of cells. The results demonstrated that miR‑34a acted as a tumor suppressor in human hepatocellular cancer cells. Following comparison of radiosensitive and radioresistant cancer cells, the results of the present study demonstrated that miR‑34a was negatively correlated with radiation resistance; and levels of miR‑34a were significantly downregulated in the HepG2 radioresistant cells. Furthermore, the rate of glycolysis in the radioresistant cells was elevated, and there was evidence that glucose uptake and lactate production increased. Lactate dehydrogenase A (LDHA), which is a key enzyme in the glycolysis signaling pathway, was found to be a target of miR‑34a in hepatocellular cancer cells. Notably, the overexpression of miR‑34a re‑sensitized HepG2 radioresistant cells to radiation treatment by inhibiting LDHA. The results of the present study revealed a negative correlation between miR‑34a and glycolysis, caused by the targeting of LDHA‑34a, providing a novel mechanism for miR‑34a‑mediated radioresistance.

Adenovirus-mediated gene therapy is a promising approach for the treatment of pancreatic cancer. We previously reported that radiation enhanced adenovirus-mediated gene expression in pancreatic cancer, suggesting that adenoviral gene therapy might be more effective in radioresistant pancreatic cancer cells. In the present study, we compared the transduction efficiency of adenovirus-delivered genes in radiosensitive and radioresistant cells, and investigated the underlying mechanisms. We used an adenovirus expressing the hepatocyte growth factor antagonist, NK4 (Ad-NK4), as a representative gene therapy. We established two radioresistant human pancreatic cancer cell lines using fractionated irradiation. Radiosensitive and radioresistant pancreatic cancer cells were infected with Ad-NK4, and NK4 levels in the cells were measured. In order to investigate the mechanisms responsible for the differences in the transduction efficiency between these cells, we measured expression of the genes mediating adenovirus infection and endocytosis. The results revealed that NK4 levels in radioresistant cells were significantly lower (P < 0.01) than those in radiosensitive cells, although there were no significant differences in adenovirus uptake between radiosensitive cells and radioresistant cells. Integrin beta3 was up-regulated and the Coxsackie virus and adenovirus receptor was down-regulated in radioresistant cells, and inhibition of integrin beta3 promoted adenovirus gene transfer. These results suggest that inhibition of integrin beta3 in radioresistant pancreatic cancer cells could enhance adenovirus-mediated gene therapy.

Identification of the genes that are differentially-expressed between radiosensitive and radioresistant cancer cells is important to the ability to predict the clinical effectiveness of radiotherapy. We established radioresistant human pancreatic cancer cell lines using fractionated irradiation in order to identify genes that are differentially-expressed between parental lines and radioresistant cell sublines. Six pancreatic cancer cell lines (PK-1, PK-8, PK-9, T3M4, MiaPaCa2 and PANC-1) were treated with 10 Gy fractionated irradiation at approximately two-week intervals (total dose 150-180 Gy). Five radioresistant sublines (PK-1, PK-8, PK-9, T3M4, and MiaPaCa2) were successfully established. Using oligonucleotide microarrays containing 17,086 genes, we identified 73 up-regulated genes and 55 down-regulated genes common to radioresistant sublines. Subsequent analysis by quantitative RT-PCR confirmed the reliability of our microarray strategy. Up-regulated genes were associated with growth factor (example, amphiregulin), cell-cycle check point (MAPKAPK2), intracellular signaling pathway (regucalcin), and angiogenesis stimulation (angiopoietin 2). Down-regulated genes were associated with apoptosis (caspase 8), retinoid esterification (lecithin retinol acyltransferase), and electron transport (calcium-activated chloride channel 1). Some of these genes have known association with response to radiation, such as caspase 8 and MAPKAPK2, but others are novel. Global gene analysis of radioresistant sublines may provide new insights into the mechanisms underlying clinical radioresistance and to improving the efficacy of radiotherapy for pancreatic cancer.

BackgroundChemo-radiotherapy, a combination of chemotherapy and radiotherapy, is the most frequent treatment for patients with esophageal cancer. In the process of radiotherapy, the radiosensitive cancer will become a radio-resistant one.MethodsIn order to detect the chemotherapeutic drug sensitivity in radio-resistant cancer cells and improve the therapy efficiency, we firstly established a radio-resistant esophageal cancer cell model (referred to as EC109/R) from the human esophageal squamous cell carcinoma cell line EC109 through fractionated irradiation using X-rays. The radio-sensitivity of EC109/R cells was measured by clonogenic assay. To detect the drug sensitivity for EC109/R compared to its parent cells, we employed MTT method to screen the effectiveness of five different drugs commonly used in clinical therapy. The ratio of apoptosis was examined by flow cytometry.ResultsEC109/R cells were more sensitive to 5-fluorouracil, doxorubicin, paclitaxel and etoposide, but tolerant to cisplatin compared to its original cells.ConclusionOur study implies that fractionated irradiation induced radio-resistant esophageal cancer cell is more sensitive to certain kind of chemotherapeutic drugs. It provides evidence for choosing the sequence of radiotherapy and chemotherapy in esophageal cancer.

Brefeldin A: A newly identified cell death inducer selectively targets radio-resistant colorectal cancer cells by direct interacting with caspase-3

Background: Radiation resistance is the main limitation of the application of radiotherapy. Ionizing radiation (IR) kills cancer cells mainly by causing DNA damage, particularly double-strand breaks (DSBs). Radioresistant cancer cells have developed the robust capability of DNA damage repair to survive IR. Nuclear factor erythroid 2-related factor 2 (NRF2) has been correlated with radiation resistance. We previously reported a novel function of NRF2 as an ATR activator in response to DSBs. However, little is known about the mechanism that how NRF2 regulates DNA damage repair and radiation resistance. Methods: The TCGA database and tissue microarray were used to analyze the correlation between NRF2 and the prognosis of lung cancer patients. The radioresistant lung cancer cells were constructed, and the role of NRF2 in radiation resistance was explored by in vivo and in vitro experiments. Immunoprecipitation, immunofluorescence and extraction of chromatin fractions were used to explore the underlying mechanisms. Results: In this study, the TCGA database and clinical lung cancer samples showed that high expression of NRF2 was associated with poor prognosis in lung cancer patients. We established radioresistant lung cancer cells expressing NRF2 at high levels, which showed increased antioxidant and DNA repair abilities. In addition, we found that NRF2 can be involved in the DNA damage response independently of its antioxidant function. Mechanistically, we demonstrated that NRF2 promoted the phosphorylation of replication protein A 32 (RPA32), and DNA topoisomerase 2-binding protein 1 (TOPBP1) was recruited to DSB sites in an NRF2-dependent manner. Conclusion: This study explored the novel role of NRF2 in promoting radiation resistance by cooperating with RPA32 and TOPBP1 to activate the ATR-CHK1 signaling pathway. In addition, the findings of this study not only provide novel insights into the molecular mechanisms underlying the radiation resistance of lung cancer cells but also validate NRF2 as a potential target for radiotherapy.