Expression of a New hemA Gene from Agrobacterium radiobacter in Escherichia coli for 5-Aminolevulinate Production

A new hemA gene encoding 5-aminolevulinate (ALA) synthase was cloned from Agrobacterium ra- diobacter zju-0121. The ALA synthase catalyzes the pyridoxal phosphate-dependent condensation of succinyl coen- zyme A (succinyl-CoA) and glycine to produce ALA. Four plasmids carrying the A, radiobacter hemA gene were transformed into different E. coli strains. The effects of both genetic and physiological factors on the expression of ALA synthase and ALA production were studied. The results indicated that the final intracellular activity of ALA synthase and the production of ALA in different expression systems varied largely. Among them, the recombinant E. coli BL21 (DE3) harboring the expression plasmid pET28-A. R-hemA was the most suitable one. The effects of isopropyl-β-D-thiogalactopyranoside (IPTG) addition time, IPTG concentration, culture temperature and the initial concentration of precursors and glucose on the ALA production were also evaluated. The expressed ALA synthase accounted for about 23.7% of the intracellular soluble protein. The highest specific activity of ALA syn- thase was 13.8nmol·min-1·mg-1 of intracellular soluble protein. In the batch culture of the recombinant E. coli, the extracellular ALA concentration reached 0.9 g·L-1.

Thymidylate synthase and thymidine phosphorylase gene expression as predictive parameters for the efficacy of 5-fluorouraci-based adjuvant chemotherapy for gastric cancer

AIM： To investigate the prognostic role of thymidylate synthase （TS） and thymidine phosphorylase （TP） mRNA levels in T3 or T4 gastric cancer treated with 5-fluorouraci-based adjuvant chemotherapy. METHODS： Fifty-one patients with T3 or T4 gastric cancer received systemic 5-fluorouraci-based adjuvant chemotherapy, and intratumoral expression of TS and TP in 51 gastric cancer tissue samples was tested by realtime quantitative PCR.RESULTS： The median disease-free survival （DFS） time was 10.2 mo in the patients. There were no significant differences in DFS between the groups with high and low levels of TP. However, the group with low level of TS had a longer DFS （14.4 mo vs 8.3 mo, P = 0.017）. The median overall survival （OS） time was 18.5 mo, and there were significant differences in OS between the groups with high and low levels of TS or TP （for TS, 17.0 mo vs 21.3 mo, P = 0.010; for TP, 16.6 mo vs 22.5 too, P = 0.009）. Moreover, the coupled low expression of these two genes was strongly associated with a longer survival time of patients as compared with that of a single gene.CONCLUSION： Expression of TS and TP mRNA is a useful predictive parameter for the survival of postoperative gastric cancer patients after 5-fluorouracilbased adjuvant chemotherapy.

The miR-9-5p/CXCL11 pathway is a key target of hydrogen sulfide-mediated inhibition of neuroinflammation in hypoxic ischemic brain injury

We previously showed that hydrogen sulfide(H2S)has a neuroprotective effect in the context of hypoxic ischemic brain injury in neonatal mice.However,the precise mechanism underlying the role of H2S in this situation remains unclear.In this study,we used a neonatal mouse model of hypoxic ischemic brain injury and a lipopolysaccharide-stimulated BV2 cell model and found that treatment with L-cysteine,a H2S precursor,attenuated the cerebral infarction and cerebral atrophy induced by hypoxia and ischemia and increased the expression of miR-9-5p and cystathionineβsynthase(a major H2S synthetase in the brain)in the prefrontal cortex.We also found that an miR-9-5p inhibitor blocked the expression of cystathionineβsynthase in the prefrontal cortex in mice with brain injury caused by hypoxia and ischemia.Furthermore,miR-9-5p overexpression increased cystathionine-β-synthase and H2S expression in the injured prefrontal cortex of mice with hypoxic ischemic brain injury.L-cysteine decreased the expression of CXCL11,an miR-9-5p target gene,in the prefrontal cortex of the mouse model and in lipopolysaccharide-stimulated BV-2 cells and increased the levels of proinflammatory cytokines BNIP3,FSTL1,SOCS2 and SOCS5,while treatment with an miR-9-5p inhibitor reversed these changes.These findings suggest that H2S can reduce neuroinflammation in a neonatal mouse model of hypoxic ischemic brain injury through regulating the miR-9-5p/CXCL11 axis and restoringβ-synthase expression,thereby playing a role in reducing neuroinflammation in hypoxic ischemic brain injury.

Herbal formula enhances 5-fluorouracil activity through suppression of thymidylate synthase

Traditional Chinese herbal medicine(TCM)has been shown to enhance the efficacy of standard anticancer agents.However,there are only a limited number of well-controlled preclinical and clinical studies documenting the potential benefit of TCM.OBJECTIVE To identify biologically active formulas that were effective against colorectal cancer(CRC)by screening TCM formulas in in vitro and in vivo animal models.METHODS Cell growth assays,cell cycle analysis,immunoblot analysis and qRT-PCR were performed to investigate the mechanism(s)of action of the formulason human CRC cells.In vivo animal models were used to evaluate the antitumor activity of formulasalone and in combination with 5-FU.RESULTS We identified Huangqin Gegen Tang(HQGGT)which suppressed the in vivo growth of human CRC HT-29 xenografts.HQGGT significantly inhibited the growth of CRC cell lines.HQGGT enhanced the cytotoxicity of 5-FU against human 5-FU-resistant cells(H630R1)and mouse colon cancer cells(MC38).This synergy was the result of suppression of thymidylate synthase expression by HQGGT.HQGGT significantly enhanced the antitumor effect of 5-FU in mice bearing MC38 xenografts.Ongoing studies have identified Huangqin as the herb responsible for TS inhibi⁃tion.CONCLUSION These findings provide support for the potential role of HQGGT as a novel modulator of fluoropyrim⁃idine chemotherapy for CRC treatment.

Catalytically Important Residues in E. coli 1-Deoxy-D-Xylulose 5-Phosphate Synthase

1-deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the initial step of the 2-C-methyl-D- erythritol 4-phosphate (MEP) pathway consisting in the condensation of (hydroxiethyl)thiamin derived from pyruvate with D-glyceraldehyde 3-phosphate (GAP) to yield 1-deoxy-D-xylulose 5-phosphate (DXP). The role of the conserved residues H49, E370, D427 and H431 of E. coli DXS was examined by site-directed mutagenesis and kinetic analysis of the purified recombinant enzyme mutants. Mutants at position H49 showed a severe reduction in their specific activities with a decrease of the kcat/KM ratio by two orders of magnitude lower than the wild-type DXS. According to available structural data residue H49 is perfectly positioned to abstract a proton from the donor substrate. Mutations in DXS E370 showed that this residue is also essential for catalytic activity. Three-dimensional structure supports its involvement in cofactor deprotonation, the first step in enzymatic thiamin catalysis. Results obtained with H431 mutant enzymes indicate that this residue plays a role contributing to transition state stabilization. Finally, mutants at position D427 also showed a severe specific activity decrease with a reduction of the kcat/KM ratio. A role in binding the substrate and selecting the stereoisomer is proposed for D427.

Improved 5-Aminolevulinic Acid Production with Recombinant Escherichia coli by a Short-term Dissolved Oxygen Shock in Fed-batch Fermentation

5-Aminolevulinic acid （ALA） is a common precursor for tetrapyrrole compounds in all kinds of organ isms and has wide applications in agriculture and medicines. In this study, a new strategy, i.e. short-term dissolved oxygen （DO） shock during aerobic fermentation, was introduced to produce 5-aminolevulinic acid with a recombi-nant E. coli. Effects of duration time of DO shock operation on plasmid concentration, intracellular ALA synthase （ALAS） activity and ALA production were investigated in Erlenmeyer shake flasks. The results indicated that both ALAS activity and ALA yield were enhanced in an anaerobic operation of 45 rain in the early exponential phase during fermentation, while they decreased when the anaerobic operation time was further increased to 60 rain. The DO shock protocol was confirmed with the fed-batch fermentation in a 15 L fermenter and the ALA production achieved 9.4 g.L-1 （72 mmol.L-1）, which is the highest yield in the fermentation broth reported up to now.

Effects of Illumination Intensity, 5-aminolevulinic Acid Concentration and Their Interaction on Chlorophyll Fluorescence Parameters and Yield of Summer Maize

[Objective] This study aimed to investigate the influence of illumination intensity, 5-aminolevulinic acid （ALA） concentration and their interaction on chlorophyll fluorescence parameters and yield of summer maize. [Method] Two illumination intensity levels and five ALA concentrations were applied in the experiment using 2x5 completely balanced program. The two illumination intensity levels were natural light （So） and 60% shade （SO, and five ALA concentrations were 0, 1, 10, 25 and 50 mg/L. [Result] The relative chlorophyll content of leaf （SPAD）, the optimal/maximal quantum yield of PS II （Fv/Fm）, the photochemical quenching coefficient （qP）, electron transport rate （ETR）, grain number per cob and grain weight per cob in $1 treatment were significantly reduced compared with that in So. However, the non- photochemical quenching coefficient （qN） was significantly increased. The responses of these parameters to ALA were different under So and $1 treatments. The SPAD, Fv/Fm, qP, ETR, grain number and grain weight per cob were firstly increased, but then decreased following the raise of ALA concentration, ranging from 0 to 50 mg/L, whereas qN showed opposite trend. The effect of the interaction of illumination in- tensity and ALA concentration on these parameters was significant （P〈0.05）. Under natural light, summer maize could obtain higher SPAD, Fv/Fm, qP and ETR and lower qN combined with low concentration of ALA. However, high concentration of ALA was needed under shading to get the same results. [Conclusion] Soaking seed in suitable concentration of ALA can reduce the yield loss of summer maize caused by short-term shading in seedling stage.

Clinical applications of 5-aminolevulinic acid-mediated fluorescence for gastric cancer

5-aminolevulinic acid(ALA) is a naturally occurring amino acid that is a protoporphyrin IX(Pp IX) precursor and a next-generation photosensitive substance. After exogenous administration of ALA, Pp IX specifically accumulates in cancer cells owing to the impaired metabolism of ALA to Pp IX in mitochondria, which results in a red fluorescence following irradiation with blue light and the formation of singlet oxygen. Fluorescence navigation by photodynamic diagnosis(PDD) using ALA provides good visualization and detection of gastric cancer lesions and is a potentially valuable diagnostic tool for gastric cancer for evaluating both the surgical resection margins and extension of the lesion. Furthermore, PDD using ALA might be used to detect peritoneal metastases during preoperative staging laparoscopy, where it could provide useful information for the selection of a therapeutic approach. Another promising application for this modality is in the evaluation of lymph node metastases. Photodynamic therapy(PDT) using ALA to cause selective damage based on the accumulation of a photosensitizer in malignant tissue is expected to be a non-invasive endoscopic treatment for superficial early gastric cancer. ALA has the potential to be used not only as a diagnostic agent but also as a therapeutic drug, resulting in a new strategy for cancer diagnosis and therapy. Here, we review the current use of PDD and PDT in gastric cancer and evaluate its future potential beyond conventional modalities combined with a light energy upconverter, a light-emitting diode and nearinfrared rays as light sources.

Recent advances in photodynamic diagnosis of gastriccancer using 5-aminolevulinic acid

Photodynamic diagnosis based on 5-aminolevulinic acidinduced protoporphyrin IX has been clinically applied in many fields based upon its evidenced efficacy and adequate safety. In order to establish a personalized medicine approach for treating gastric cancer patients, rapid intraoperative detection of malignant lesions has become important. Feasibility of photodynamic diagnosis using 5-aminolevulinic acid for gastric cancer patients has been investigated, especially for the detection of peritoneal dissemination and lymph node metastasis. This method enables intraoperative realtime fluorescence detection of peritoneal dissemination, exhibiting higher sensitivity than white light observation without histopathological examination. The method also enables detection of metastatic foci within excised lymph nodes, exhibiting a diagnostic accuracy comparable to that of a current molecular diagnostics technique. Although several complicating issues still need to be resolved, such as the effect of tissue autofluorescence and the insufficient depth penetration of excitation light, this simple and rapid method has the potential to become a useful diagnostic tool for gastric cancer, as well as urinary bladder cancer and glioma.

Effects of Exogenous 5-Aminolevulinic Acid and 24-Epibrassinolide on Cd Accumulation in Rice from Cd-Contaminated Soil

High grain-Cd-accumulating rice variety Yongyou 9 was planted in Cd-contaminated farmland in Taizhou City, Zhejiang Province, China to study the effects of 5-aminolevulinic acid(ALA) and24-epibrassinolide(EBR) on Cd accumulation in brown rice. Results showed that the exogenous ALA and EBR had no significant effects on agronomic traits, soil pH and total Cd content in soil, but had some effects on the available Cd content in soil, and significantly influenced the Cd accumulation in the different parts of rice. Results also showed that 100 mg/L exogenous ALA significantly reduced the Cd accumulation in brown rice to blow the food safety standard(0.2 mg/kg), and also significantly reduced the Cd contents in the roots and culm of rice. However, 200 mg/L exogenous ALA treatment increased the Cd content in brown rice remarkably. In addition, 0.15 mg/L EBR treatment increased Cd accumulation in roots, culm, leaves and brown rice notably, whereas 0.30 mg/L exogenous EBR treatment reduced the Cd accumulation in brown rice properly, but it was not significant. Therefore,proper concentration of ALA can effectively reduce the Cd accumulation in brown rice, which can be used as an effective technical method for the safe production of rice in Cd polluted farmland.

Immunohistochemical expression of thymidylate synthase and prognosis in gastric cancer patients submitted to fluoropyrimidine-based chemotherapy

Objective： Adjuvant chemotherapy with 5-fluorouracil （5-FU） has been widely used in gastric cancer （GC） patients to prevent relapse after curative resection. 5-FU acts by inhibiting thymidylate synthase （TS）, and high levels of TS correlate with resistance to treatment with fluoropyfimidines. The aim of this study was to evaluate the expression of TS in GC patients, and its relation with clinicopathological characteristics and prognosis in adjuvant chemotherapy with 5-FU. Methods： We retrospectively evaluated 285 patients who underwent D2-gastrectomy with curative intent. TS expression was determined by immunohistochemistry （IHC） in tumor cells by tissue microarray （TMA）. TS level was evaluated according to the intensity and percentage of cells marked by a score system. Patients were divided in three groups according to their TS-score： negative, low and high. Results： TS expression was positive in 92.3% of GC. TS-high, TS-low and TS-negative were observed in 46.3%, 46.0% and 7.7% of patients, respectively. High-TS GC were associated with older age （P=0.007）, high neutrophil/lymphocyte ratio （P=0.048）, well/moderately differentiated histology （P=0.001）, intestinal Lauren type （P〈0.001） and absence of perineural invasion （P=0.003）. Among 285 patients, 133 stage IUIII patients （46.7%） received chemotherapy with 5-FU. In survival analysis, TS-high was associated with worse disease-free survival （DFS） in stage III GC patients who received 5-FU-based chemotherapy （P=0.007）. Multivariate analysis revealed that total gastrectomy, poorly differentiated tumors and high TS-score were associated with worse DFS in stage III GC patients. Conclusions： High TS-score in stage III GC was associated with poor DFS in patients treated with fluoropyrimidine-based chemotherapy.

Insight into 5-aminolevulinic acid-induced modulation of cellular antioxidant metabolism to confer salinity and drought tolerance in maize

The current study investigated the comparative oxidative damage in twomaize seedlings induced by saline,drought,and combined stress and the ameliorative role of two different doses(20 and 80μM)of 5-aminolevulinic acid(ALA)against the above-mentioned stresses.Hydroponically grown 10-day-old maize(Zea mays,var.BARI Hybrid Maize-7(BHM-7)and BARI Hybrid Maize-9(BHM-9))seedlings were exposed to 12 dS/m of saline solution,200 mM mannitol-induced drought stress alone and their combined stress for 7 days.Result revealed that individual stresses retard the plant growth to some degrees,however,their combined stress has more detrimental effects,which might be correlated with lipid peroxidation(MDA)-induced oxidative stress in seedlings,enhanced Na+/K+ratio,and augmented generation of superoxide(O_(2)•^(−))and hydrogen peroxide(H_(2)O_(2)).In contrast,exogenous ALA supplementation at 20μM concentration markedly recovered from chlorosis and growth inhibition,substantially scavenged reactive oxygen species(ROS)and MDA by preserving ionhomeostasis and relaxing oxidative stress,also,by boosting catalase(CAT)and glutathione S-transferase(GST),and exclusively via depressing the activity of lipoxygenase(LOX)antioxidant enzyme.On the contrary,80μM ALA made things worse,nevertheless,higher activities shown by other antioxidant enzymes,like,superoxide dismutase(SOD),ascorbate peroxidase(APX),peroxidase(POD),and glutathione peroxidase(GPX),which were related to lessen the oxidative damage by highly produced O_(2)•^(−)and H_(2)O_(2) under combined stress.Non-denaturing gel electrophoresis was done for further confirmation.However,ALA importantly increased the photosynthetic pigment contents in both genotypes irrespective of doses.Nevertheless,GST might have assisted the plants to escape from the herbicidal effect by detoxification.However,in the combined stress condition,high ALA concentration may have some positive role to play.Our findings also showed that BHM-9 performed better than BHM-7.Therefore,ALA at lower concentration was effective for single stress of saline and drought,while higher concentration can improve plant survival under combined stress.

Study on Insecticidal Activities and Effect on Three Kinds of Enzymes by 5-Aminolevulinic Acid on Oxya chinensis

Insecticidal activities and effects on three enzymic activities caused by 5-aminolevulinic acid （ALA） on Oxya chinensis were studied. Fourth-instar nymphs of O. chinensis were treated with different doses ofALA （A1,250 mM; A2, 450 mM; A3,750 mM; A4, 1 000 mM）. Mortality and the activities of acetylcholinesterase （ACHE）, glutathione S-transferase （GSTs）, and glutathione peroxidase （GPx） were determinated. The mortality of O. chinensis rose with an increasing dose of ALA. The mortality of high-dose treatments A3 and A4 reached 66.19 and 80.21%, respectively. The value of LD50 was 3.61 （3.29-3.93） mg·g^-1 body weight （95% confidence interval）. Biochemical studies showed that the activities of AChE and GPx in the A4 treatment declined by 51.53 and 42.82% in the female, and 42.65 and 43.85% in the male compared to the control, respectively, and the degree of decline reached a significant level at P 〈 0.05. Meanwhile, the GSTs activities of O. chinensis enhanced with increasing dose of ALA. The GSTs activities of female and male O. chinensis in the A4 treatment remarkably increased by 171.05 and 97.42% compared to the control （P〈 0.05）. ALA had an obviously toxic effect on O. chinensis. Moreover, ALA caused the photoinactivation of AChE and GPx, which induced nerve transmission blocking and the capability to defend oxidation damage declining. Meanwhile, a high dose of ALA could activate GSTs, which caused a feedback inhibition of the insect to the phototoxic substance.

A Photosensitivity Insecticide, 5-Aminolevulinic Acid, Exerts EffectiveToxicity to Oxya chinensis (Orthoptera: Acridoidea)

5-Aminolevulinic acid （ALA）, a major photosensitivity insecticide, has attracted increasing attention as a new type of highly efficient, environmental friendly pesticide to be used to control the pest. To examine whether or not ALA acts effectively to grasshopper, Oxya chinensis and elucidate the detoxification mechanism of ALA, the susceptibility to ALA was assessed in O. chinensis and two major metabolic detoxification enzymes including glutathione S-transferases （GSTs） and general esterases （ESTs）-specific activities were compared in different development stages and different body sections of O. chinensis treated by ALA and the control. The results showed that the ALA exhibited obvious toxicity to the grasshopper in different development stages. In the low-dose treatment （0.0597 mmol L-1）, the mortalities of O. chinensis reached a significant level （55.5% in the 1st instar nymphs, 61.4% in the 2nd instar nymphs, 71.4% in the 3rd instar nymphs, and 64.4% in the 4th instar nymphs. But, there was no dose-dependent toxic effect. Thereby, we proposed that ALA has the potential for acting as photosensitivity insecticide for controlling O. chinensis. GSTs activity assays using CDNB and DCNB as substrates indicated that the thorax and abdomen of the different instar nymphs treated by ALA showed 1.52-5.56 fold significantly increased GSTs activities compared with the control. However, for the ESTs-specific activity assay, there was no significant difference between O. chinensis treated by ALA and the control within different instar nymphs, when a-NA, a-NB and b-NA were used as substrates. Therefore, GSTs-mediated metabolic detoxification as evidenced by significantly increased GSTs activities might contribute to protect against oxidative damage and oxidative stress by ALA in O. chinensis.

The Role of 5-Aminolevulinic Acid (5-ALA) and Sleep

Objective: To determine if there is a relationship between the administration of the dietary supplement containing 5-Aminolevulinic Acid (5-ALA) and sleep. Methods: A double-blind, randomized parallel-group study was conducted. It was a 4-month study of 40 participants between the ages of 40 and 70. Males and females were recruited equally. There were 20 in each group who had existing sleep disorders. The tool used to measure participant sleep improvement included the Pittsburgh Insomnia Rating Scale-20 Question (PIRS-20). Improved sleep is reflected when the total PIRS-20 score is lower. Results: Improvement in sleep in the group taking 50 mg 5-ALA, compared to controls, was significant. The mean change, from baseline through week 6, was -5.67 units less on the sleep scale than the control group with a p value of 0.001. The mean change from week 6 to week 10 when the participant was no longer taking the supplement was 4.55 units higher than the control group with a p value of 0.062, which is of borderline significance. Conclusions: There is a relationship between the administration of dietary supplements containing 5-ALA and sleep. The results of this study suggest that 5-ALA is associated with improved sleep. The mechanism for sleep improvement needs to be explored. Further research is warranted.

Development of a Photodynamic Diagnosis Method for Oral Squamous Cell Carcinoma Using 5-Aminolevulinic Acid and a Luminescence Plate Reader

Purpose: To establish a simple and accurate photodynamic diagnosis (PDD) method for oral squamous cell carcinoma (OSCC). Methods: OSCC cell lines HSC-2, HSC-3, HSC-4, and Sa3, and normal human oral keratinocytes (HOK) were used. First, we examined the amount of cells needed to detect differences in fluorescence intensities for PDD. OSCC cell lines were adjusted to concentrations of 1 × 104 (104), 1 × 105 (105), and 1 × 106 (106) cells/ml. The experimental groups comprised a group with 5-aminolevulinic acid (5-ALA (+)), and a group without 5-ALA (5-ALA (-)). For each OSCC cell line, 100 μl of each concentration of cells of the 5-ALA groups was seeded onto fluorescence plates, and fluorescence intensity was measured at 60-min intervals for 240 min. Results are expressed as the ratio of fluorescence intensity in 5-ALA (+) to 5-ALA (-). As cells at the concentration of 106 cells/ml provided the clearest results, fluorescence intensities of all cell lines were measured using this concentration at 20-min intervals for 700 min using the same methods. Results: The 5-ALA (+) to (-) ratio increased in a cell concentration-dependent manner at 240 min;the ratio was highest with 106 cells/ml and lowest with 104 cells/ml. With 106 cells/ml in the 5-ALA (+) group, fluorescence intensity increased in a metabolic time-dependent manner;the increase was highest in HSC-2 cells, followed by HSC-4 cells, HSC-3 cells, Sa3 cells, and HOK. Fluorescence intensity was significantly enhanced after 40 min in HSC-2, HSC-3, and HSC-4 cells, after 60 min in Sa3 cells, and after 100 min in HOK compared to the 5-ALA (-) group (P < 0.05). Moreover, fluorescence intensity was significantly increased in OSCC cell lines compared to HOK after 40 min. Conclusion: Early detection of OSCC is possible by screening only microplate reader measurements of fluorescence intensity for PDD.

吡咯啉-5-羧酸合成酶在人肝癌组织中表达及其对人肝癌细胞系铁死亡的调控作用观察

目的 观察吡咯啉-5-羧酸合成酶(Pyrroline-5-carboxylate synthase,P5CS)在肝细胞癌(Hepatocellular car-cinoma,HCC)组织中的表达变化及其对人肝癌细胞系铁死亡的调控作用。方法 (1)HCC及癌旁组织P5CS检测:选择10例HCC患者的癌组织及癌旁组织,采用免疫组化法检测HCC及癌旁组织P5CS蛋白;(2)P5CS对人肝癌细胞系Li-7、Huh-7铁死亡的调控作用观察:取对数生长期Li-7、Huh-7,用si ALDH18A1[乙醛脱氢酶家族18成员A1(AL-DH18A1)可编码P5CS蛋白]转染细胞,采用q RT-PCR法检测转染前后Li-7及Huh-7细胞脂质过氧化物合成相关基因ACSL4、LPCAT3、LOX-15,采用WESTERN Blotting法检测转染前后Li-7及Huh-7细胞铁死亡相关蛋白溶质载体家族7成员11(SLC7A11)、谷胱甘肽过氧化物酶(GPX4)。取转染前后Li-7及Huh-7细胞各分为2组,分别加入5μmol/L的铁死亡诱导剂Erastin、同体积DMSO,采用C11-BODIPY荧光探针检测各组细胞脂质过氧化物含量。取转染前、后Li-7及Huh-7细胞各分为2组,分别加入5μmol/L的GPX4靶向抑制剂RSL-3、5μmol/L的Erastin、同体积DMSO,采用C11-BODIPY荧光探针检测各组细胞脂质过氧化物含量。结果 HCC及癌旁组织P5CS蛋白相对表达量分别为6.80±2.10、0.40±0.20,二者比较,P<0.05。与转染前比较,转染后Li-7及Huh-7细胞SLC7A11、GPX4蛋白相对表达量低(P均<0.05)。与转染前比较,转染后加入Erastin、RSL-3的Li-7、Huh-7细胞脂质过氧化物含量均升高(P均<0.05)。与加入Erastin者比较,转染后加入RSL-3的Li-7、Huh-7细胞脂质过氧化物含量高、细胞活性低(P均<0.05)。结论 HCC组织中P5CS表达升高。P5CS可抑制Li-7及Huh-7细胞的铁死亡,P5CS可能通过抑制细胞GPX4表达,调控Li-7及Huh-7细胞的铁死亡。

Photodynamic Therapy of Cancer Cells with Methanesulfonate Salts of 5-Aminolevulinic Acid and Its Derivatives

A series of 5-aminolevulinic acid and its alkylester methanesulfonates was exploited to photodynamic therapy（PDT） of human lymphocytic cells, U-937 in vitro. The PDT efficiency is influenced by the concentration and incubation time. Generally, for ALA and its alkylester methanesulfonates, the cell survival rate decreases and the accumulation ability of PplX increases with the concentration and incubation time. We found that the longer carbon chain methanesulfonates（C5-S, C6-S, C8-S） exhibit better PDT effect than ALA methanesulfonate. This possibly provides a promising route to the clinical application ofPplX-mediated PDT to cancer cell.

5-氟尿嘧啶耐药在结直肠癌治疗中的研究进展

结直肠癌(CRC)是全球最常见的癌症之一,发病率和死亡率都很高。5-氟尿嘧啶(5-FU)是CRC患者化疗的一线推荐药物,也是用于治疗CRC的基石药物。随着对其作用机制了解的增加,在过去的几十年中,已经开发了各种治疗方式以增加其抗癌活性。但CRC仍具有较高的发病率,化疗耐药仍是CRC治疗的主要障碍之一。结论:在本文中,我们系统地回顾和更新了5-FU耐药机理和其与氟尿嘧啶联合用药治疗结直肠癌的现有知识。因此本文就氟尿嘧啶耐药在结直肠癌治疗中的研究进展进行综述。