Heavy ion irradiation inhibits in vitro angiogenesis even at sublethal dose

Angiogenesis is essential for tumor growth and metastasis. Because endothelial cells are genetically stable, they rarely acquire resistance to anticancer modalities, and could, thus, be a suitable target for radiation therapy. Heavy ion radiation therapy has attracted attention as an effective modality for cancer therapy because of its highly lethal effects, but the effects of heavy ion irradiation on in vitro cell function associated with angiogenesis have not been reported. Our study found that in vitro angiogenesis was inhibited by high linear energy transfer carbon ion irradiation even at sublethal dose (0.1 Gy). ECV304 and HUVEC human umbilical vascular endothelial cells were irradiated with 290 MeV carbon ion beams of approximately 110 keV/ micro m or 4 MV X-ray of approximately 1 keV/ micro m. Their adhesiveness and migration to vitronectin or osteopontin were inhibited, and capillary-like tube structures in three-dimensional culture were destroyed after carbon ion irradiation concomitant with the inhibition of matrix metalloproteinase-2 activity, down-regulation of alphaVbeta3 integrin, which is one of the adhesion molecules, slight up-regulation of membrane type1- matrix metalloproteinase, and significant up-regulation of tissue inhibitor of metalloproteinase-2. On the other hand, sublethal X-ray irradiation promoted migration of endothelial cells, and the capillary-like tube structure in three-dimensional culture progressed even after 16 Gy irradiation. These results provide an implication that heavy ion beam therapy could be superior to conventional photon beam therapy in preventive effects on in vitro angiogenesis even at sublethal dose, and might inhibit angiogenesis in vivo.     

Sublethal irradiation promotes migration and invasiveness of glioma cells: implications for radiotherapy of human glioblastoma

Human malignant gliomas are highly lethal neoplasms. Involved-field radiotherapy is the most important therapeutic measure. Most relapses originate from the close vicinity of the irradiated target field. Here, we report that sublethal doses of irradiation enhance the migration and invasiveness of human malignant glioma cells. This hitherto unknown biological effect of irradiation is p53 independent, involves enhanced alphavbeta3 integrin expression, an altered profile of matrix metalloproteinase-2 and matrix metalloproteinase-9 (MMP-2 and MMP-9) expression and activity, altered membrane type 1 MMP and tissue inhibitor of metalloproteinases-2 expression, and an altered BCL-2/BAX rheostat favoring resistance to apoptosis. BCL-2 gene transfer and irradiation cooperate to enhance migration and invasiveness in a synergistic manner. Sublethal irradiation of rat 9L glioma cells results in the formation of a greater number of tumor satellites in the rat brain in vivo concomitant with enhanced MMP-2 and reduced tissue inhibitor of metalloproteinases-2 expression. Collectively, these data suggest that the current concepts of involved-field radiotherapy for malignant glioma need to be reconsidered and that the pharmacological inhibition of migration and invasion during radiotherapy may represent a new therapeutic approach to improve the therapeutic efficacy of radiotherapy for malignant glioma.     

Radiation-induced increase in invasive potential of human pancreatic cancer cells and its blockade by a matrix metalloproteinase inhibitor, CGS27023.

PURPOSE: Radiotherapy remains a major therapeutic option for patients with advanced pancreatic cancer. Nevertheless, the effects of irradiation on malignant biological behaviors (e.g., migration and invasion of cancer cells) have yet to be clarified. Thus, we conducted an in vitro study to investigate the radiation-induced alterations around cell migration and invasion capacity. EXPERIMENT DESIGN: Three cell lines from human pancreatic cancer were included in the study. gamma-radiation was used for irradiation treatment. Cell migration and invasion ability were evaluated by Transwell migration assay and Matrigel invasion assay. The activity of MMP-2 and 9, and expression of urokinase-type plasminogen activator were investigated with gelatin zymography and immunoblot, respectively. RESULTS: Irradiation enhances invasive potential in some pancreatic cancer cells, whereas it significantly inhibits cell proliferation and migration. This hitherto unknown biological effect of irradiation involves enhanced matrix metalloproteinase (MMP)-2 activity. Consequently, simultaneous administration of an MMP inhibitor, CGS27023A, suppresses the radiation-enhanced invasion through blockade of transition of MMP-2 from latent type to active type. CONCLUSION: Because radiation may increase invasion ability through activating MMP proteolytic system, simultaneous administration of the MMP inhibitor during radiotherapy could be a potent adjuvant therapeutic approach to improve the efficacy of radiotherapy for pancreatic cancer.     

Carbon-ion radiation enhances migration ability and invasiveness of the pancreatic cancer cell, PANC-1, in vitro

Pancreatic cancer is an aggressive disease that responds poorly to conventional photon radiotherapy. Carbon-ion (C-ion) radiation has advantages compared with conventional radiotherapy, because it enables more accurate dose distribution and more efficient tumor cell killing. To elucidate the effects of local radiotherapy on the characteristics of metastatic tumors, it is necessary to understand the nature of motility in irradiated tumor cells; this will, in turn, facilitate the development of effective strategies to counter tumor cell motility, which can be used in combination with radiotherapy. The aim of the present study was to examine the invasiveness of pancreatic cancer cells exposed to C-ion irradiation. We found that C-ion irradiation suppressed the migration of MIAPaCa-2, BxPC-3 and AsPC-1; diminished the invasiveness of MIAPaCa-2; and tended to reduce the invasion of BxPC-3 and AsPC-1. However, C-ion irradiation increased the invasiveness of PANC-1 through the activation of plasmin and urokinase-type plasiminogen activator. Administration of serine protease inhibitor (SerPI) alone failed to reduce C-ion-induced PANC-1 invasiveness, whereas the combination of SerPI and Rho-associated coiled-coil forming protein kinase (ROCK) inhibitor suppressed it. Furthermore, PANC-1 showed mesenchymal-amoeboid transition when we treated with SerPI alone. In conclusion, C-ion irradiation is effective in suppressing the invasive potential of several pancreatic tumor cell lines, but not PANC-1; this is the first study showing that C-ion irradiation induces the invasive potential of a tumor cell line. Further in vivo studies are required to examine the therapeutic effectiveness of radiotherapy combined with inhibitors of both mesenchymal and amoeboid modes of tumor cell motility.     

成人胶质瘤质子与重离子治疗现状及进展

脑胶质瘤是最常见的原发脑恶性肿瘤,标准治疗是手术联合术后放疗,但疗效并不理想。质子和碳离子治疗是目前最先进的放疗技术,在多种肿瘤中显示出优于光子放疗疗效和生存质量。然而,质子与碳离子治疗应用于胶质瘤的作用尚未明确,本文将就质子与碳离子治疗成人胶质瘤的基础研究和临床结果进行详细阐述。     

Preclinical biological assessment of proton and carbon ion beams at Hyogo Ion Beam Medical Center

PURPOSE: To assess the biologic effects of proton and carbon ion beams before clinical use. METHODS AND MATERIALS: Cultured cells from human salivary gland cancer (HSG cells) were irradiated at 5 points along a 190 MeV per nucleon proton and a 320 MeV per nucleon carbon ion beam, with Bragg peaks modulated to 6 cm widths. A linac 4 MV X-ray was used as a reference. Relative biologic effectiveness (RBE) values at each point were calculated from survival curves. Cells were also irradiated in a cell-stack phantom to identify that localized cell deaths were observed at predefined depth. Total body irradiation of C3H/He mice was performed, and the number of regenerating crypts per jejunal section was compared to calculate intestinal RBE values. For carbon ion and referential 4 MV X-ray beams, mouse right legs were irradiated by four-fractional treatment and followed up for skin reaction scoring. RESULTS: RBE values calculated from cell survival curves at the dose that would reduce cell survival to 10% (D10) ranged from 1.01 to 1.05 for protons and from 1.23 to 2.56 for carbon ions. The cell-stack phantom irradiation revealed localized cell deaths at predefined depth. The intestinal RBE values ranged from 1.01 to 1.08 for protons and from 1.15 to 1.88 for carbon ions. The skin RBE value was 2.16 at C320/6 cm spread-out Bragg peak (SOBP) center. CONCLUSION: The radiobiologic measurements of proton and carbon ion beams at Hyogo Ion Beam Medical Center are consistent with previous reports using proton beams in clinical settings and carbon ion beams with similar linear energy transfer (LET) values.     

Biological effectiveness and relative biological effectiveness of ion beams for in‐vitro cell irradiation

Biological effectiveness and relative biological effectiveness are critical for proton and ion beam radiotherapy. However, the relationship between the two quantities and physical character of ion beams is not well established. By analyzing 1188 sets of in‐vitro cell irradiation experiments using ion beams ranging from protons to ²³⁸U, compiled by the Particle Irradiation Data Ensemble (PIDE) project, the biological effectiveness of the ion beams, with cell survival fractionation (SF) as the endpoint, was found to be dependent on the fluence and linear energy transfer (LET) of the ion beam. Consequently, the relative biological effectiveness of the ion beam to photon beam was also established as a function of LET. A common form of relationship among SF, fluence, and LET was found to be valid for all ion beam experiments. The close form relationship could be used for proton and ion beam radiotherapy applications.     

RAB5A对人肺腺癌细胞系侵袭转移作用的研究

目的：探讨RAB5A基因在人肺腺癌细胞系侵袭和转移中的作用。方法：采用体外重建基底膜侵袭实验和癌细胞粘附能力；癌细胞趋化性运动能力、癌细胞分泌明胶酶的能力及活性的测定，分析RAB5A正义真核表达载体转染后的AGZY83－a和反义RNA转染后的Anip973细胞的侵袭、转移能力的改变。结果：RAB5A正义表达载体PcDNA3.1-RAB5A转染的AGZY83－a重建基底膜侵袭能力明显增强，统计学意义显著（P＜0．0005），细胞趋化性运动能力显著增高（P＜0．0005），对基底膜成分的粘附能力增大（P＜0．05），以及明胶酶分泌及活性增强等一系列趋向Anip973的变化。PcDNA3-AntiRAB5A反义RNA表达载体对Anip973重建基底膜侵袭力明显下降（P＜0．0025），趋化性运动能力显著降低（P＜0．005），对基底膜成分粘附能力降低（P＜0．05），以及明胶酶分泌减少等一系列趋向AGZY83－a的变化。结论：RAB5A在肺腺癌侵袭轩移表型形成中发挥重要的作用。反义RNA可阻断RAB5A基因的翻译过程。     

Proton beam therapy for inoperable recurrence of bronchial high-grade mucoepidermoid carcinoma

We report the case of a 17-year-old patient who received four courses of proton beam therapy for inoperable recurrent high-grade bronchial mucoepidermoid carcinoma of the chest wall and lymph nodes. The equivalent doses in conventional fractionation of 79.2-80.6 Gy were applied to the tumor from the first to third courses of proton beam therapy; the hemi-chest wall was also irradiated prophylactically in the third course. The irradiated tumor recurred marginally and liver metastasis developed, but tumor size within the irradiated field was suppressed. Proton beam therapy was also applied to the marginally recurrent tumor in the fourth course. The patient died of cancer about 5 years after the first course of proton beam therapy-about 9 years after the initial diagnosis and surgery. Repeated irradiation of the mediastinum and chest wall with photon radiotherapy is often limited by side-effects in the heart, esophagus and spinal cord. However, no severe late complications in critical organs were detected in this case. Only a Grade 2 skin reaction and lymphatic edema were observed. Therefore, high-dose proton beam therapy may be an option as a salvage therapy with less toxicity to normal tissues compared with photon radiotherapy and provide an alternative to repeated surgery.     

Irradiated C6 glioma cells induce angiogenesis in vivo and activate endothelial cells in vitro

Malignant gliomas are angiogenesis dependent and present a remarkable degree of resistance to radiotherapy. In the present work, we studied the effect of irradiation of C6 glioma cells on their proliferation and activation in vitro and on glioma cell-induced angiogenesis in vivo and in vitro. Irradiation of C6 glioma cells decreased cell proliferation in a dose-dependent manner. Interestingly, metalloproteinase-2 and -9 expression and secretion, as well as integrin alpha(v) expression, increased with elevated doses of X rays 48 hr after irradiation and was mostly evident at the higher doses used. When pre-irradiated C6 cells were implanted on nonirradiated chicken embryo chorioallantoic membranes (CAMs), there was a significant dose-dependent increase in tumor induced angiogenesis, compared to angiogenesis induced by nonirradiated cells. Similar results were obtained when C6 cells were irradiated 48 hr after their inoculation onto nonirradiated CAMs. In the same line, conditioned medium from irradiated C6 cells significantly increased endothelial cell proliferation and migration in vitro, in a manner dependent on the dose of X rays. These results explain at least in part the low effectiveness of radiation therapy of malignant gliomas and support the notion that inhibition of angiogenesis in parallel with radiotherapy may represent a new therapeutic approach.     

Mesenchymal stem cells are resistant to carbon ion radiotherapy

Mesenchymal stem cells (MSCs) participate in regeneration of tissues damaged by ionizing radiation. However, radiation can damage MSCs themselves.Here we show that cellular morphology, adhesion and migration abilities were not measurably altered by photon or carbon ion irradiation. The potential for differentiation was unaffected by either form of radiation, and established MSC surface markers were found to be stably expressed irrespective of radiation treatment. MSCs were able to efficiently repair DNA double strand breaks induced by both high-dose photon and carbon ion radiation. We have shown for the first time that MSCs are relatively resistant to therapeutic carbon ion radiotherapy. Additionally, this form of radiation did not markedly alter the defining stem cell properties or the expression of established surface markers in MSCs.     

Elevation of seprase expression and promotion of an invasive phenotype by collagenous matrices in ovarian tumor cells

Tumor cells do not constitutively exhibit invasive activity, but rather, can be transiently induced to adhere and form lesions. We report here that the expression of seprase, a dominant EDTA-resistant gelatinase in malignant tumors, is dependent on tumor cell exposure to type I collagen gel (TICg). The induced seprase expression of ovarian tumor cells influences their collagen contraction and invasion capability. Importantly, tumor cells with reduced seprase expression, due to manipulation by RNA interference, showed a reduction of TICg contraction in the gel contractility assay, inhibition of tumor cell invasion through TICg as shown by a transwell migration assay and inhibition of peritoneal membrane tumor lesion in a mouse model. In addition, mAb C27, an antibody against beta1 integrin, which blocks cellular avidity to TICg, can induce seprase RNA expression and promote the invasive phenotype and metastatic potential of ovarian tumor cells. Thus, collagenous matrices in the tumor cell niche induce the expression of seprase and initiate tumor invasion and metastatic cascades.     

Effects of carbon ion beam on putative colon cancer stem cells and its comparison with X-rays

Although carbon ion therapy facilities are expensive, the biological effects of carbon ion beam treatment may be better against cancer (and cancer stem cells) than the effects of a photon beam. To investigate whether a carbon ion beam may have a biological advantage over X-rays by targeting cancer stem-like cells, human colon cancer cells were used in vitro and in vivo. The in vitro relative biological effectiveness (RBE) values of a carbon ion beam relative to X-rays at the D10 values were from 1.63 to 1.74. Cancer stem-like CD133(+), CD44(+)/ESA(+) cells had a greater ability for colony and spheroid formation, as well as in vivo tumorigenicity compared with the CD133(-), CD44(-)/ESA(-) cells. FACS (fluorescence-activated cell sorting) data showed that cancer stem-like cells were more highly enriched after irradiation with X-rays than carbon ion at doses that produced the same level of biological efficacy. A colony assay for cancer stem-like cells showed that RBE values calculated by the D10 levels were from 2.05 to 2.28 for the carbon ion beam relative to X-rays. The in vivo xenotransplant assay showed an RBE of 3.05 to 3.25, calculated from the slope of the dose-response curve for tumor growth suppression. Carbon ion irradiation with 15 Gy induced more severe xenograft tumor cell cavitation and fibrosis without significant enhancement of cells with putative cancer stem cell markers, CD133, ESA, and CD44, compared with 30 Gy X-rays, and marker positive cells were significantly decreased following 30 Gy carbon ion irradiation. Taken together, carbon ion beam therapy may have an advantage over photon beam therapy by improved targeting of putative colon cancer stem-like cells.     

七氟烷对乳腺癌BT549细胞侵袭、迁移及MMP-2、MMP-9表达的影响

目的:探讨七氟烷对乳腺癌BT549细胞侵袭、迁移及基质金属蛋白酶-2（MMP-2）、基质金属蛋白酶-9（MMP-9）表达的影响。方法:乳腺癌BT549细胞用七氟烷处理后,细胞划痕实验检测细胞迁移,Transwell小室检测细胞侵袭,Western blot检测MMP-2、MMP-9蛋白表达。结果:七氟烷处理后的BT549细胞迁移率由（36.45±3.21）%降低至（14.68±1.52）%,侵袭细胞数目由（136.58±13.36）个减少到（79.95±14.25）个,MMP-2水平由0.48±0.04减少为0.14±0.03,MMP-9水平由0.96±0.07减少为0.22±0.05,两组比较差异均具有统计学意义（P<0.05）。结论:七氟烷能够抑制乳腺癌BT549细胞迁移和侵袭,降低细胞中MMP-2、MMP-9表达水平。     

重离子辐照对人舌鳞癌CAL27细胞的生物学效应及其分子机制

目的 研究重离子12C6+离子辐照对人舌鳞癌CAL27细胞的生物学效应以及分子机制。方法 应用不同剂量重离子束（12C6+）对体外培养的人舌癌CAL27细胞进行辐照,采用MTT、Transwell、流式细胞术、划痕实验、克隆实验观察重离子束（12C6+）对CAL27细胞迁移和侵袭、凋亡和周期的影响,Western blot法观察重离子束（12C6+）对CAL27细胞增殖的影响。结果 （1）重离子辐照对CAL27细胞的增殖抑制作用与剂量-时间成正相关;（2）和空白对照组比较,在相同时间点,不同剂量的重离子束（12C6+）对细胞中P53、P65和VEGF的表达影响明显,随着重离子束（12C6+）辐照剂量增大,细胞中P53、P65和VEGF的表达明显降低（P<0.05）。结论 重离子辐照能抑制CAL27细胞的增殖,且具有剂量和时间效应。     

Manganese superoxide dismutase enhances the invasive and migratory activity of tumor cells

Clinically significant elevations in the expression of manganese superoxide dismutase (Sod2) are associated with an increased frequency of tumor invasion and metastasis in certain cancers. The aim of this study was to examine whether increases in Sod2 activity modulate the migratory potential of tumor cells, contributing to their enhanced metastatic behavior. Overexpression of Sod2 in HT-1080 fibrosarcoma cells significantly enhanced their migration 2-fold in a wound healing assay and their invasive potential 3-fold in a transwell invasion assay. Severity of invasion was directly correlated to Sod2 expression levels and this invasive phenotype was similarly observed in 253J bladder tumor cells, in which Sod expression resulted in a 3-fold increase in invasion compared with controls. Further, migration and invasion of the Sod2-expressing cells was inhibited following overexpression of catalase, indicating that the promigratory/invasive phenotype of Sod2-expressing cells is H(2)O(2) dependent. Sod2 overexpression was associated with a loss of vinculin-positive focal adhesions that were recovered in cells coexpressing catalase. Tail vein injections of Sod2-GFP-expressing HT-1080 cells in NCR nude mice led to the development of pulmonary metastatic nodules displaying high Sod2-GFP expression. Isolated tumors were shown to retain high Sod2 activity in culture and elevated levels of the matrix degrading protein matrix metalloproteinase-1, and a promigratory phenotype was observed in a population of cells growing out from the tumor nodule. These findings suggest that the association between increased Sod2 activity and poor prognosis in cancer can be attributed to alterations in their migratory and invasive capacity.     

Overcoming hypoxia-induced tumor radioresistance in non-small cell lung cancer by targeting DNA-dependent protein kinase in combination with carbon ion irradiation

Background

Hypoxia-induced radioresistance constitutes a major obstacle for a curative treatment of cancer. The aim of this study was to investigate effects of photon and carbon ion irradiation in combination with inhibitors of DNA-Damage Response (DDR) on tumor cell radiosensitivity under hypoxic conditions.

Methods

Human non-small cell lung cancer (NSCLC) models, A549 and H1437, were irradiated with dose series of photon and carbon ions under hypoxia (1% O₂) vs. normoxic conditions (21% O₂). Clonogenic survival was studied after dual combinations of radiotherapy with inhibitors of DNA-dependent Protein Kinase (DNAPKi, M3814) and ATM serine/threonine kinase (ATMi).

Results

The OER at 30% survival for photon irradiation of A549 cells was 1.4. The maximal oxygen effect measured as survival ratio was 2.34 at 8 Gy photon irradiation of A549 cells. In contrast, no significant oxygen effect was found after carbon ion irradiation. Accordingly, the relative effect of 6 Gy carbon ions was determined as 3.8 under normoxia and. 4.11 under hypoxia. ATM and DNA-PK inhibitors dose dependently sensitized tumor cells for both radiation qualities. For 100 nM DNAPKi the survival ratio at 4 Gy more than doubled from 1.59 under normoxia to 3.3 under hypoxia revealing a strong radiosensitizing effect under hypoxic conditions. In contrast, this ratio only moderately increased after photon irradiation and ATMi under hypoxia. The most effective treatment was combined carbon ion irradiation and DNA damage repair inhibition.

Conclusions

Carbon ions efficiently eradicate hypoxic tumor cells. Both, ATMi and DNAPKi elicit radiosensitizing effects. DNAPKi preferentially sensitizes hypoxic cells to radiotherapy.