Combination of chemotherapy and radiotherapy. II. Differential effect on the medullary syndrome of irradiation combined with the drug administered in fractional doses and at various times]

We have studied the effect of 5-Fu in fractional doses on the medullar syndrome after a single whole body cobalt irradiation in mice. The bone marrow is not sensitive to the fractionation of 5-Fu and it is the single dose of 5-Fu injected 72 hours after the irradiation which is the most effective.     

Effect of salt solutions on radiosensitivity of mammalian cells. III. Treatment with hypertonic solutions.

V79 Chinese hamster cells were treated with hypertonic solutions of NaCl or KCl and irradiated rat various times before, during, or after exposure to the solution. In solutions of molarities between 0-2 and 0-5 M, the cellular radiosensitivity increases with the molarity of the bathing solution. At these molarities, the hypertonic solution need not be present during irradiation to sensitize cells. Furthermore, radiosensitivity of cells could be increased by exposing cells for longer times to the hypertonic solution before irradiation. At higher salt concentrations (at 1-5 to 1-8 M), significant radioprotection is observed. Survival curve data showed that this protection was characterized by an increase in DO and a decrease in n, while the survival curves of cells sensitized with 0-465 M NaCl or with lower concentrations exhibited mainly changes in DO. The 1-55 M NaCl solution must be present during radiation to give a protective effect. Prolonged exposure to the salt before irradiation reduced the amount of radioprotection afforded by the salt. The results are discussed in terms of the effects of ions on histones, cellular water structure and the cell-aging cycle.     

Antioxidants decrease the apoptotic effect of 5-Fu in colon cancer by regulating Src-dependent caspase-7 phosphorylation

Although the rate of development of drug resistance remains very high, 5-fluorouracil (5-Fu) is still the most common chemotherapeutic drug used for the treatment of colon cancer. A better understanding of the mechanism of why cancers develop resistance to 5-Fu could improve its therapeutic effect. Sometimes, antioxidants are used simultaneously with 5-Fu treatment. However, a recent clinical trial showed no advantage or even a harmful effect of combining antioxidants with 5-Fu compared with administration of 5-Fu alone. The mechanism explaining this phenomenon is still poorly understood. In this study, we show that 5-Fu can induce reactive oxygen species-dependent Src activation in colon cancer cells. Mouse embryonic fibroblasts that are deficient in Src showed a clear resistance to 5-Fu, and knocking down Src protein expression in colon cancer cells also decreased 5-Fu-induced apoptosis. We found that Src could interact with and phosphorylate caspase-7 at multiple tyrosine sites. Functionally, the tyrosine phosphorylation of caspase-7 increases its activity, thereby enhancing cellular apoptosis. When using 5-Fu and antioxidants together, Src activation was blocked, resulting in decreased 5-Fu-induced apoptosis. Our results provide a novel explanation as to why 5-Fu is not effective in combination with some antioxidants in colon cancer patients, which is important for clinical chemotherapy.     

PUMA在结肠癌细胞中的表达及诱导细胞凋亡中的作用

目的:通过5-Fu诱导携带有野生型p53基因的HCT116和携带有突变性p53基因的HT-29两种结肠癌细胞系,比较两株细胞在各时间点凋亡水平和PUMA mRNA表达情况的差异,探讨PUMA对细胞凋亡的作用及诱导其表达的基本途径。方法:用逆转录聚合酶链反应(RT—PCR)检测不同结肠癌细胞株HT-29、HCT116在抗肿瘤药物5-Fu作用下不同时间点结肠癌细胞株内PUMA mRNA表达水平的差异,用吖啶橙/溴化乙啶(AO/EB)荧光染色检测各时间点细胞的凋亡水平,分析其与PUMAmRNA表达水平之间的关系。结果:携带有野生型P53基因的结肠癌细胞株HCT116在5-Fu作用下6h即可出现PUMA mRNA的表达,随着药物作用时间的延长其表达强度增加,并且与细胞凋亡水平呈正相关;含有突变型P53基因的结肠癌细胞株HT-29在5-Fu作用下无PUMA的表达。结论:通过5-Fu诱导细胞凋亡出现的PUMA表达需要野生型P53基因,突变型P53基因无法诱导PUMA的表达。PUMA与结肠癌细胞凋亡水平呈正相关,是一种促凋亡蛋白。     

Reactions of the intracellular NADpool in the yeastS. cerevisiae after UV-C- or X-ray irradiation

Summary The reaction of the intracellular NADpool after irradiation of cells either with UV-C light or with X-rays was studied in four different strains of the yeastS. cerevisiae. We found neither in wildtype strains nor in radiation sensitive mutants remarkable changes in the NADpool within 2 h after irradiation. Preculture of cells in medium enriched with nicotinic acid, a precursor of NAD, influenced the intracellular NAD concentration only to a small extend in all strains, but enhanced the radiation resistance against UV-C significantly in one rad6 mutant strain. The uptake of NAD and NAC by all strains before and after irradiation with UV-C and X-ray was tested also. NAD generally is taken up by the cells to a very low extent before and after irradiation without irradiation-dose dependency. NAC is taken up by all strains before and after irradiation. Only the rad6 mutant exhibited an irradiation-dose dependent NAC-uptake after UV-C irradiation.Abbreviations ADPRT ADPribosyltransferase - NAC nicotinic acid - YEPG yeast extract-peptone-glucose medium     

Interactions of drugs and radiation in haemopoietic tissue assessed by lethality of mice after whole-body irradiation

Drug-radiation interactions in haemopoietic tissue were assessed as the lethality of mice within 7-28 days after whole-body irradiation. The investigated drugs were adriamycin (ADM), bleomycin (BLM), cyclophosphamide (CTX), 5-fluorouracil (5-FU), methotrexate (MTX), mitomycin C (MM-C) and cis-diamminedichloroplatinum II (cis-DDP). The drugs were administered as single doses 15 min before graded doses of whole-body irradiation or at different intervals from 7 days before to 7 days after fixed radiation doses. ADM, CTX, 5-FU, MM-C and cis-DDP enhanced the radiation response when administered 15 min before irradiation. The dose effect factor (DEF) was 9.11 for 5-FU and in the range 1.25-1.59 for the other drugs. MTX administration 15 min before irradiation had no effect (DEF 1.00). However, MTX increased lethality if given 1-3 days after irradiation (DEF 1.21-1.76) and protected against lethality if given 1-3 days before irradiation (DEF 0.83). A similar time dependence was observed for ADM, CTX, 5-FU, MM-C and cis-DDP. Protection against lethality was not observed but in all these cases the lethality was significantly lower at administration 1-3 days before than 1-3 days after irradiation. A proper investigation of the effect of BLM was not possible as the combination of this drug and whole-body irradiation caused a high rate of gastrointestinal deaths.     

The effect of He-Ne laser radiation on the survival of HeLa cells subjected to ionizing radiation]

A monolayer of HeLa cells, at the stationary phase of growth, exposed to He-Ne laser radiation (632.8 nm; 100 J/m2) either 5 min or 60 min prior to gamma irradiation (0.1-10 Gy; 6.75 Gy/min), or 5 min after irradiation has been investigated. With a 5-min interval between irradiation sessions (both sequences) the survival curves are virtually the same as those for gamma-irradiated cells only. With He-Ne laser radiation delivered 60 min before gamma irradiation with doses exceeding 5 Gy, a fraction of radioresistant cells is identified whose D0 is almost twice as high as D0 of basic cell mass (3.6 and 1.7 Gy respectively. The survival curve becomes a two-component one. A hypothesis is proposed that He-Ne laser radiation activates, in some cells, the processes that promote the repair of radiation damages.     

5-Fluorouracil induced dysregulation of the microbiome-gut-brain axis manifesting as depressive like behaviors in rats

Disturbances of the gut microbiome have been widely suggested to be associated with 5-fluorouracil (5-Fu) induced digestive pathologies. Furthermore, it has been elucidated that the gut microbiome may play a key role in the pathogenesis of depressive disorders via the microbiota-gut-brain axis. Despite the speculation, there exists no direct evidence proving the causality between disturbances in the gut microbiome induced by 5-Fu and depressive mood dysregulation. Herein, behavioral testing was used to evaluate depressive-like behaviors in 5-Fu treated rats. Subsequently, the gut microbiota and prefrontal cortex (PFC) metabolic were analyzed by 16S rRNA sequencing and ¹H nuclear magnetic resonance (¹H NMR). To clarify the association between the gut microbiota and their role on depressive-like behaviors caused by 5-Fu, a fecal microbiota transplantation (FMT) experiment was carried out. The results suggested that 5-Fu could significantly alter the diversity and abundance of the gut microbiome, and induce PFC metabolic disorders, as well as depressive behaviors in rats. Transplantation of fecal microbiota from healthy control into 5-Fu treated rats significantly alleviated the PFC metabolic disorder and depressive-like behaviors. In conclusion, this study demonstrated that the gut microbiome was actively involved in the occurrence of 5-Fu induced depressive-like behaviors, and manipulation of specific gut microbiome parameters may serve as a promising novel target for side effects of 5-Fu treatment.     

Combination chemotherapy and radiotherapy. Differential effect, on the medullary syndrome, of fractionated irradiation with fractionated drug administration

The study of the different effects of 5 FU administration before and after fractional irradiation on the medullar syndrome show the importance of the schedule in the effectiveness of the administration of the gamma rays and the cytostatic. As for the intestinal syndrome, the best results were obtained when 5 FU is administrated in one single dose per week 9 hours after the fifth irradiation for a two weeks schedule.     

低强度532nm与633nm激光血管内照射生物效应比较

目的：研究同等照射条件的低强度532nm与633nm激光血管内照射对家兔白细胞计数与淋巴细胞凋亡的影响,比较两种激光生物效应的特点。方法：用532nm和633nm激光对健康日本大耳白家兔血管内照射,平均照射功率均设在5mW左右,照射总能量约12J。两组家兔均于照前及照后1d、4d、7d、11d进行外周血白细胞计数,于照前及照后1d、5d进行淋巴细胞凋亡分析。结果：532nm激光照射后,家兔外周血白细胞计数表现为先显著升高后趋向恢复,633nm激光照射后白细胞计数变化类似,但与照前相比升高不明显;与照前相比,两组家免外周血淋巴细胞凋亡比例于照后1d均明显降低,照后5d均显著升高;两组家兔相比,照射后白细胞计数差别明显,但淋巴细胞凋亡比例差异不显著。结论：同等照射条件下,低强度532nm与633nm激光照射血液的生物效应相似,都可以促进白细胞的代谢更新,只是532nm激光的效应略强一些。     

The effect of autoblood in a hyposmotic state on the colony-forming activity of hematopoietic stem cells]

It has been shown that hypoosmotic autoblood injected sub- or intracutaneously stimulates the colony-forming activity of haemopoietic stem cells in mice. Autoblood injected to animals immediately after their irradiation stimulates haemopoiesis even after a single dose. When mice are injected with autoblood prior to irradiation, the time between the first injection and the day of irradiation is critical for manifestation of the immunomodulating effect. Autoblood infusions immediately before, the day before, or two days before irradiation markedly deteriorate the clinical status of experimental animals and cause death in some of them. It is suggested that stimulation of haemopoiesis is associated with the appearance in the blood stream of a population of radiosensitive cells, apparently T-cell precursors.     

Tegafur Substitution for 5-Fu in Combination with Actinomycin D to Treat Gestational Trophoblastic Neoplasm

Although 5-fluorouracil (5-Fu) combination chemotherapy provides a satisfactory therapeutic response in patients with gestational trophoblastic neoplasms (GTNs), it has severe side effects. The current study analyzed the therapeutic effects and side effects of tegafur plus actinomycin D (Act-D) vs. 5-Fu plus Act-D for the treatment of GTNs based on controlled historical records. A total of 427 GTN cases that received tegafur and Act-D combination chemotherapy at the Second Xiangya Hospital of XiangYa Medical School between August 2003 and July 2013 were analyzed based on historical data. A total of 393 GTN cases that received 5-Fu plus Act-D between August 1993 and July 2003 at the same hospital were also analyzed, which constituted the control group. The therapeutic effects, toxicity and side effects after chemotherapy were compared between the groups. The overall response rate was 90.63% in the tegafur+Act-D group (tegafur group) and 92.37% in the 5-Fu+Act-D group (5-Fu group); these rates were not significantly different (P > 0.05). However, the incidence rates of myelosuppression (white blood cell decline), gastrointestinal reactions (nausea, vomiting, dental ulcer, and diarrhea), skin lesions and phlebitis were lower in the tegafur group than in the 5-Fu group (P < 0.05). The results of this study may provide useful data for the clinical application of tegafur in GTN treatment.     

Gypenosides Synergistically Enhances the Anti-Tumor Effect of 5-Fluorouracil on Colorectal Cancer In Vitro and In Vivo: A Role for Oxidative Stress-Mediated DNA Damage and p53 Activation

Objective

5-Fluorouracil (5-Fu) has been widely used as a first-line drug for colorectal cancer (CRC) treatment but limited by drug resistance and severe toxicity. The chemo-sensitizers that augment its efficiency and overcome its limitation are urgently needed. Gypenosides (Gyp), the main components from Gynostemma pentaphyllum (Thunb.) Makino, has shown potential anti-tumor property with little side-effect. Here, we carefully explored the chemo-sensitization of Gyp to potentiate the anti-tumor effect of 5-Fu in vitro and in vivo.

Methodology / Principal Findings

3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltertrazolium bromide tetrazolium assay and colony formation test reveal that Gyp could significantly enhance the 5-Fu-caused SW-480,SW-620 and Caco2 cells viability loss. Calcusyn analysis shows that Gyp acts synergistically with 5-Fu. Annexin V-PE/7-AAD staining indicates 5-Fu + Gyp could induce SW-480 cell apoptosis. The activations of caspase 3, caspase 9 and poly (ADP-ribose) polymerase (PARP) were involved in the process. Gyp was also found to up-regulate 5-Fu-caused phospho-p53 expression and thus augment 5-Fu-induced G0/G1 phase arrest. Gyp elevated intracellular ROS level, significantly enhanced 5-Fu-triggered DNA damage response as evidenced by flow cytometry, comet assay and the expression of Ser139-Histone H2A.X. Inhibition of ROS and p53 respectively reversed the cell death induced by 5-Fu + Gyp, suggesting the key roles of ROS and p53 in the process. Moreover, 5-Fu and Gyp in combination exhibits much superior tumor volume and weight inhibition on CT-26 xenograft mouse model in comparison to 5-Fu or Gyp alone. Immunohistochemistry analysis suggests the combinations greatly suppressed tumor proliferation. Preliminary toxicological results show that 5-Fu + Gyp treatment is relatively safe.

Conclusions

As a potential chemo-sensitizer, Gyp displays a splendid synergistic effect with 5-Fu to inhibit cancer cell proliferation and tumor growth. By using 5-Fu and Gyp in combination would be a promising therapeutic strategy for CRC treatment.     

Phytochrome and Seed Germination. III. Action of Prolonged Far Red Irradiation on the Germination of Tomato and Cucumber Seeds

Prolonged irradiation with continuous or intermittent far red prevents the germination of tomato and cucumber seeds. The inhibitory efficiency of intermittent far red decreases with the lengthening of the interval between successive irradiations, and with the increase of temperature. If each far red irradiation is followed by red, germination is restored. Intermittent far red is less inhibitory than intermittent red-far red when red is given immediately before each far red. This effect is more evident when the interval between successive irradiation becomes longer.     

Booster irradiation to the spleen following total body irradiation. A new immunosuppressive approach for allogeneic bone marrow transplantation

Graft rejection presents a major obstacle for transplantation of T cell-depleted bone marrow in HLA-mismatched patients. In a primate model, after conditioning exactly as for leukemia patients, it was shown that over 99% of the residual host clonable T cells are concentrated in the spleen on day 5 after completion of cytoreduction. We have now corroborated these findings in a mouse model. After 9-Gy total body irradiation (TBI), the total number of Thy-1.2+ cells in the spleen reaches a peak between days 3 and 4 after TBI. The T cell population is composed of both L3T4 (helper) and Lyt-2 (suppressor) T cells, the former being the major subpopulation. Specific booster irradiation to the spleen (5 Gy twice) on days 2 and 4 after TBI greatly enhances production of donor-type chimera after transplantation of T cell-depleted allogeneic bone marrow. Similar enhancement can be achieved by splenectomy on day 3 or 4 after TBI but not if splenectomy is performed 1 day before TBI or 1 day after TBI, strengthening the hypothesis that, after lethal TBI in mice, the remaining host T cells migrate from the periphery to the spleen. These results suggest that a delayed booster irradiation to the spleen may be beneficial as an additional immunosuppressive agent in the conditioning of leukemia patients, in order to reduce the incidence of bone marrow allograft rejection.     

Postirradiation sensitization of mammalian cells by the thiol-depleting agent dimethyl fumarate

Dimethyl fumarate (DMF) depletes intracellular glutathione (GSH) by covalent bond formation in a reaction mediated by GSH-S-transferase. Treatment of hypoxic Chinese hamster V79 cells with 5 mM DMF before irradiation radiosensitizes the cells, resulting in an enhancement ratio (ER) of about 2.7 with minimal toxicity, when the end point is clonogenic cell survival. Under the same conditions aerobic cells are sensitized, and ER of about 1.3 is found, and GSH is reduced to about 3% of control. Very similar results were obtained previously with Chinese hamster ovary (CHO) cells. In addition, new data presented here show that DMF treatment of V79 or CHO cells immediately after irradiation under hypoxic conditions sensitizes the cells, resulting in an ER of about 1.5, DMF treatment after irradiation under aerobic conditions results in an ER of 1.3, and this DMF treatment reduces protein thiols (PSH) to about 70% of control. When induction of DNA damage is measured using the neutral elution assay, treatment of V79 or CHO cells with DMF prior to irradiation under hypoxic conditions results in an ER of 1.9-2.0, but there is no enhancement of DNA damage when DMF is added after irradiation under hypoxic conditions or when cells are treated with DMF before or after irradiation under aerobic conditions. Based on these data we postulate that DMF radiosensitizes killing of hypoxic cells by two actions: depletion of GSH interferes with the chemical competition between damage fixation and repair, and depletion of PSH causes an inhibition of enzymatic repair processes. We also suggest that DMF sensitizes aerobic cells only by inhibition of enzymatic repair processes.     

The action of caffeine on X-irradiated HeLa cells. VIII. Recovery from potentially lethal damage

Recovery from potentially lethal radiation damage in HeLa S3 cells has been studied by irradiating synchronous cultures with 4 Gy at selected ages in the cell cycle, initiating treatment with 4 mM caffeine, which prevents recovery, at progressively later times up to 24-30 h after irradiation, and determining the plateau level of survival after incubation with the caffeine until 36-40 h after mitotic collection. Cell recovery appears to begin immediately after irradiation at any time during interphase: an accelerating increase in survival gives way after several hours to a linear increase which lasts for an additional several hours. The median recovery time is approximately 13 h after irradiation at any time during G1, but is markedly shorter (5-7 h) after irradiation in S or G2. The rate of recovery is slightly depressed if DNA replication is inhibited with aphidicolin after irradiation and slightly enhanced if protein synthesis is inhibited with cycloheximide. Both the rate and the extent of recovery are dependent on the location of the cells in the cycle at the time of irradiation--both functions increasing with cell age from the beginning of S, but having different age dependencies in G1. Blocking cell progression with a DNA-synthesis inhibitor before irradiation halts the age-dependent changes.