Increased glucose influx and glycogenesis in lung cancer cells surviving after irradiation.

PURPOSE: Lung cancer is considered as one of the most frequent malignancies worldwide. Radiotherapy is the main treatment modality applied for locally advanced disease, but remnant surviving cancer tissue results in disease progression in the majority of irradiated lung carcinomas. Metabolic reprogramming is regarded as a cancer hallmark and is associated with resistance to radiation therapy. Here, we explored metabolic alterations possibly related to cancer cell radioresistance. MATERIALS AND METHODS: We compared the expression of metabolism-related enzymes in the parental A549 lung cancer cell line along with two new cell lines derived from A549 cells after recovery from three (A549-IR3) and six (A549-IR6) irradiation doses with 4 Gy. Differential GLUT1 and GYS1 expression on proliferation and radioresistance were also comparatively investigated. RESULTS: A549-IR cells displayed increased extracellular glucose absorption, and enhanced mRNA and protein levels of the GLUT1 glucose transporter. GLUT1 inhibition with BAY-876, suppressed cell proliferation and the effect was significantly more profound on A549-IR3 cells. Protein levels of molecules associated with aerobic or anaerobic glycolysis, or the phosphate pentose pathway were similar in all three cell lines. However, glycogen synthase 1 (GYS1) was upregulated, especially in the A549-IR3 cell line, suggestive of glycogen accumulation in cells surviving post irradiation. GYS1-gene silencing repressed the proliferation capacity of A549, but this increased their radioresistance. The radio-protective effect of the suppression of proliferative activity induced by GYS1 silencing did not protect A549-IR3 cells against further irradiation. CONCLUSIONS: These findings indicate that GYS1 activity is a critical component of the metabolism of lung cancer cells surviving after fractionated radiotherapy. Targeting the glycogen metabolic reprogramming after irradiation may be a valuable approach to pursue eradication of the post-radiotherapy remnant of disease.

Lipophagy-Related Protein Perilipin-3 and Resistance of Prostate Cancer to Radiation Therapy.

PURPOSE: Radiation therapy is a principal treatment modality for localized and locally advanced prostate cancer (PCa). Metabolic alterations, including lipid metabolism, may reduce treatment efficacy, resulting in tumor relapse and poor therapeutic outcome. In the current study, we investigated the role of the lipophagy-related protein perilipin-3 (PLIN3) and the lysosomal acid lipase (LAL) in PCa response to radiation therapy. METHODS AND MATERIALS: We explored the in vitro and xenograft (in NOD SCID and R2G2 mice) response to radiation of either PLIN3-depleted or LAL-depleted hormone-refractory (DU145, PC3) and hormone-responsive (22Rv1) PCa cell lines. Moreover, we evaluated the clinical role of PLIN3 and LAL protein expression in a series of PCa tissue specimens from patients treated with radical radiation therapy. RESULTS: In vitro and in vivo experiments showed reduced proliferation and strong radiosensitization of all studied PCa cell lines upon PLIN3 depletion. In vivo experiments demonstrated the significantly augmented radiation therapy efficacy upon PLIN3 depletion, resulting in extensive tissue necrosis. Overexpression of PLIN3 in tissue specimens was correlated with an increased MIB1 proliferation index, increased autophagy flux, reduced response to radiation therapy, and poor prognosis. The effect of LAL depletion on radiation therapy was of lesser importance. CONCLUSIONS: Assessment of PLIN3 expression may identify subgroups of patients with PCa who are less responsive to radiation therapy and at high risk of relapse after irradiation. Whether radiation therapy efficacy may be enhanced by concurrent autophagy or PLIN3 inhibition in this subgroup of patients demands clinical evaluation.

Apalutamide radio-sensitisation of prostate cancer.

INTRODUCTION: The combination of radiotherapy with bicalutamide is the standard treatment of prostate cancer patients with high-risk or locally advanced disease. Whether new-generation anti-androgens, like apalutamide, can improve the radio-curability of these patients is an emerging challenge. MATERIALS AND METHODS: We comparatively examined the radio-sensitising activity of apalutamide and bicalutamide in hormone-sensitive (22Rv1) and hormone-resistant (PC3, DU145) prostate cancer cell lines. Experiments with xenografts were performed for the 22Rv1 cell line. RESULTS: Radiation dose-response viability and clonogenic assays showed that apalutamide had a stronger radio-sensitising activity for all three cell lines. Confocal imaging for γΗ2Αx showed similar DNA double-strand break repair kinetics for apalutamide and bicalutamide. No difference was noted in the apoptotic pathway. A striking cell death pattern involving nuclear karyorrhexis and cell pyknosis in the G1/S phase was exclusively noted when radiation was combined with apalutamide. In vivo experiments in SCID and R2G2 mice showed significantly higher efficacy of radiotherapy (2 and 4 Gy) when combined with apalutamide, resulting in extensive xenograft necrosis. CONCLUSIONS: In vitro and in vivo experiments support the superiority of apalutamide over bicalutamide in combination with radiotherapy in prostate cancer. Clinical studies are encouraged to show whether replacement of bicalutamide with apalutamide may improve the curability rates.

Suppressed PLIN3 frequently occurs in prostate cancer, promoting docetaxel resistance via intensified autophagy, an event reversed by chloroquine.

Lipid metabolism reprogramming is one of the adaptive events that drive tumor development and survival, and may account for resistance to chemotherapeutic drugs. Perilipins are structural proteins associated with lipophagy and lipid droplet integrity, and their overexpression is associated with tumor aggressiveness. Here, we sought to explore the role of lipid droplet-related protein perilipin-3 (PLIN3) in prostate cancer (PCa) chemotherapy. We investigated the role of PLIN3 suppression in docetaxel cytotoxic activity in PCa cell lines. Additional effects of PLIN3 depletion on autophagy-related proteins and gene expression patterns, apoptotic potential, proliferation rate, and ATP levels were examined. Depletion of PLIN3 resulted in docetaxel resistance, accompanied by enhanced autophagic flux. We further assessed the synergistic effect of autophagy suppression with chloroquine on docetaxel cytotoxicity. Inhibition of autophagy with chloroquine reversed chemoresistance of stably transfected shPLIN3 PCa cell lines, with no effect on the parental ones. The shPLIN3 cell lines also exhibited reduced Caspase-9 related apoptosis initiation. Moreover, we assessed PLIN3 expression in a series of PCa tissue specimens, were complete or partial loss of PLIN3 expression was frequently noted in 70% of the evaluated specimens. Following PLIN3 silencing, PCa cells were characterized by impaired lipophagy and acquired an enhanced autophagic response upon docetaxel-induced cytotoxic stress. Such an adaptation leads to resistance to docetaxel, which could be reversed by the autophagy blocker chloroquine. Given the frequent loss of PLIN3 expression in PCa specimens, we suggest that combination of docetaxel with chloroquine may improve the efficacy of docetaxel treatment in PLIN3-deficient cancer patients.

'Stemness' and 'senescence' related escape pathways are dose dependent in lung cancer cells surviving post irradiation.

AIMS: Lung cancer is one of the main causes of cancer-related deaths worldwide and radiotherapy is a major treatment of choice. However, radioresistance is a main reason for radiotherapy failure or tumor relapse. Here, we investigated possible mechanisms associated with cancer cell radioresistance. MATERIALS AND METHODS: We compared two newly derived cell lines, namely A549-IR3 and A549-IR6, which survived repeated (3 or 6 times) 4 Gy exposure of parental A549 lung cancer cell line. DNA repair ability, stemness and senescence were comparatively studied. KEY FINDINGS: A549-IR3 exhibited higher proliferation ability and radioresistance compared to parental and A549-IR6 cells. Enhanced radioresistance was not accompanied by chemoresistance to cisplatin or docetaxel. DNA repair kinetics (γΗ2ΑΧ expression) were similar in all cell lines. A549-IR3 cells exhibited a significant rise in stem cell markers (CD44, CD133, OCT4, SOX2 and NANOG) whereas A549-IR6 displayed an increased senescent population. SIGNIFICANCE: Cancer cells surviving after radiotherapy may follow two different escape pathways: selection for radioresistance resulting in regrowth, and in clinical terms relapse, or above an irradiation threshold, stem-cells die and cancer cells become senescent, leading the tumor to a state of dormancy.

Blocking LDHA glycolytic pathway sensitizes glioblastoma cells to radiation and temozolomide.

PURPOSE: Up-regulation of lactate dehydrogenase LDHA, is a frequent event in human malignancies and relate to poor postoperative outcome. In the current study we examined the hypothesis that LDHA and anaerobic glycolysis, may contribute to the resistance of glioblastoma to radiotherapy and to temozolomide. METHODS AND MATERIALS: The expression of LDH5 isoenzyme (fully encoded by the LDHA gene) was assessed in human glioblastoma tissues. Experimental in vitro studies involved the T98 and U87 glioblastoma cell lines. Their sensitivity to radiotherapy and to temozolomide, following silencing of LDHA gene or following exposure to the LDHA chemical inhibitor 'oxamate' and to the glycolysis inhibitor '2-deoxy-d-glucose' (2DG), was studied. RESULTS: Glioblastoma tissues showed strong cytoplasmic and nuclear LDH5 expression in 0-90% (median 20%) of the neoplastic cells. T98 and U87 cell lines showed that blocking glycolysis, either with LDHA gene silencing or exposure to oxamate (30 mM) and blockage of glycolysis with 2DG (500 µM), results in enhanced radiation sensitivity, an effect that was more robust in the T98 radioresistant cell line. Furthermore, all three glycolysis targeting methods, significantly sensitized both cell lines to Temozolomide. CONCLUSIONS: The current study provides evidence that a large subgroup of human glioblastomas are highly glycolytic, and that inhibitors of glycolysis, like LDHA targeting agents, may prove of therapeutic importance by enhancing the efficacy of radiotherapy and temozolomide against this lethal disease.