Nutritional support in the treatment of aplastic anemia.

OBJECTIVE: Whether a specific nutritional support promotes healing of aplastic anemia (AA) patients is still unclear. Therefore, we explored the potential of a high-nucleotide, arginine, and micronutrient nutritional supplement on the nutritional rehabilitation of AA mice. METHODS: The BALB/c AA mice model was treated with hypodermic injections of acetylphenylhydrazine (100 mg/kg), x-ray (2.0 Gy), and intraperitoneal injections of a cyclophosphamide (80 mg/kg) combination. Then AA mice were fed with nutritional supplements in a dose-dependent manner (1445.55, 963.7, 674.59 mg/kg/d) for 7 wk. At the end of the experimental period, mice were autopsied. A full blood count was performed, and femoral marrow cell suspensions were prepared to assess the total femoral nucleated cell count and the number of committed hemopoietic progenitor cells (colony-forming units). The pathologic changes of liver and spleen were analyzed. RESULTS: The significant increases of nutrient mixture groups were evident in many peripheral blood parameters. The femoral nucleated cell count and colony-forming units of nutritional supplements groups were markedly increased, compared with the AA group. Transmission electron microscopy showed that the number of mitochondria in similar bone marrow cells was increased in nutritional supplements groups. The nutritional supplements also affected the recovery of livers and spleens of AA mice. CONCLUSION: Specific nutritional supplements accelerated rehabilitation of AA mice and can be used as nutritional support in the treatment of AA.

Nutritional rehabilitation of mitochondrial aberrations in aplastic anaemia.

Aplastic anaemia (AA) is a disease characterised by bone marrow hypocellularity and peripheral blood pancytopenia. AA is also associated with mitochondrial aberrations. The present study was undertaken primarily to test the hypothesis that a nutrient mixture could affect the nutritional rehabilitation of mitochondrial aberrations in AA mice. BALB/c AA mice were induced by a combination of hypodermic injections of acetylphenylhydrazine (100 mg/kg), X-rays (2·0 Gy) and intraperitoneal injections of cyclophosphamide (80 mg/kg). We treated these mice with nutrient mixture-supplemented diets in a dose-dependent manner (1445·55, 963·7, 674·59 mg/kg per d), and the effects of the nutrient mixture for mitochondrial rehabilitation were analysed in AA mice. Transmission electron microscopy showed that mitochondrial ultrastructural abnormalities in bone marrow cells, splenocytes and hepatocytes of the nutrient mixture groups were restored markedly, compared with the AA group. Mitochondrial membrane potentials of the nutrient mixture groups were increased remarkably. Western blot analysis also revealed that the nutrient mixture significantly inhibited cytochrome c release of mitochondria in the AA group. Furthermore, the mitochondrial DNA content of the nutrient mixture groups was also increased. Our data suggest that the nutrient mixture may promote the rehabilitation of mitochondrial aberrations, and consequently protects against mitochondrial dysfunction in AA mice.

Hepatocyte growth factor induces invasion and migration of ovarian cancer cells by decreasing the expression of E-cadherin, beta-catenin, and caveolin-1.

Distant metastases are unusual occurrences at presentation and during the progression of epithelial ovarian cancer. There are no good clinical predictors of this phenomenon. In this study, we examined the role of hepatocyte growth factor (HGF) in cell metastasis in ovarian cancer HO8910 cells. We found that HGF has functions in biological processes essential to metastasis, including morphological remodeling, invasion and migration (P = 0.000, P = 0.001). Western blotting showed that in HGF treated group, the expression of E-cadherin, beta-catenin, and caveolin-1 was decreased as compared with the control group (P = 0.000, P = 0.002, P = 0.000). Immunofluorescence staining demonstrated that the population of E-cadherin, beta-catenin, and caveolin-1 at the cell membrane was downregulated. Reverse transcriptase polymerase chain reaction analysis revealed the decreased expression of E-cadherin, beta-catenin, and caveolin-1 at the mRNA level. Our data indicated that HGF leads to downregulation of E-cadherin, beta-catenin, and caveolin-1, disassembly of cell-cell contacts, and invasion and migration enhancement in human ovarian cancer cells.

Reversal effect of Raf-1/Mdr-1 siRNAs co-transfection on multidrug resistance in KBv200 cell line.

Multidrug resistance (MDR) is a major barrier for chemotherapy of many cancers. Mdr-1 plays a key role in the development of MDR as extensively verified. However, the role of Raf-1 overexpression in the development of multidrug resistance in human squamous carcinoma (KBv200) cells remains largely unknown. The aim of this study was to investigate the correlation of Raf-1 overexpression with the development of multidrug resistance in KBv200 cells. Furthermore, we explored the reversal effect of Raf-1 siRNA transfection and Raf-1/Mdr-1 siRNAs co-transfection on the multidrug resistance of KBv200 cells and potential mechanism of reversing the multidrug resistance. MTT and flow cytometry assay were used to investigate the reversal effect of single transfection with either Raf-1 or Mdr-1 siRNA and double transfection with Raf-1/Mdr-1 siRNAs to vincristine of KBv200 cells. RT-PCR, immunofluorescence and Western Blot were used to detect mRNA and protein expression of Raf-1 and multidrug-resistant gene Mdr-1. The results of gene detection showed that the expression levels of both Raf-1 and Mdr-1 were greatly decreased upon Raf-1 silencing alone or in combination with Mdr-1 silencing. Raf-1 or Mdr-1 siRNA single transfection could reverse the multidrug resistance of KBv200 cells effectively. Compared with single transfection, Raf-1/Mdr-1 siRNAs co-transfection can significantly reduce IC(50) values and increase the apoptotic rates of KBv200 cells. The above results suggested that Raf-1 gene may be a novel target for reversing the multidrug resistance of human squamous carcinoma cells. Raf-1/Mdr-1 siRNAs co-transfection might be a promising approach to abrogate the multidrug resistance of cancer cells. The potential mechanism may be via inhibiting the multidrug-resistant gene Mdr-1 expression efficiently.

[Growth-inhibiting effect of psoralen plus ultraviolet-A light therapy on K562 cells].

OBJECTIVE: To observe the effects of psoralen plus ultraviolet-A light (PUVA) on K562 cells and the relative mechanism. METHODS: The effects of psoralen, ultraviolet-A light and PUVA on K562 cells were assayed by monotetrazolium test (MTT). DNA content was analyzed by flow cytometry (FCM). The apoptotic rates of K562 cells treated with 40 and 80 microg/ml psoralen for 24 and 48 hours were assayed by Annexin-V-FITC/PI reagent kit on FCM respectively. The ultrastructures of apoptotic cells were observed by a transmission electron microscope (TEM). RESULTS: Either single psoralen therapy or single ultraviolet-A irradiation had inhibiting effect on K562 cells. The inhibiting effect of PUVA on K562 cells was stronger than that of the single psoralen therapy or single ultraviolet-A light irradiation (P<0.05). Apoptotic peak (AP) was detected by FCM. TEM test showed that K562 cells treated with PUVA were smaller, having condensed cell nucleus, assembled chromatin, disintegrated nucleus body and the majority of the cells appeared to be apoptotic conformation. CONCLUSION: Psoralen has inhibiting effect on K562 cells, and the effect of PUVA is more significant. It is suggested that 10 min irradiation and 40 microg/ml terminal concentration of psoralen be probably the best choice for PUVA. The inhibiting effect of PUVA is due to apoptosis.

[Expression of aurora-A kinase in human lung cancer cell lines PG, A549, and NCI-H460].

BACKGROUND & OBJECTIVE: Recent researches found that Aurora-A overexpresses in various malignancies. This study was to detect the expression of Aurora-A in lung cancer cell lines PG (highly-metastatic giant cell lung cancer), A549 (lung adenocarcinoma), and NCI-H460 (large cell lung cancer) and explore its correlation to DNA content, provide a theoretical basis for screening tumor marker and molecular therapeutic target of lung cancer. METHODS: mRNA and protein levels of Aurora-A in PG, A549, and NCI-H460 cells were detected by reverse transcription-polymerase chain reaction(RT-PCR) and Western blot. Flow cytometry was used to analyze DNA contents in cell cycles of PG, A549, and NCI-H460 cells. RESULTS: mRNA level of Aurora-A was 1.14 in PG cells, 1.16 in A549 cells, and 0.84 in NCI-H460 cells, respectively; protein level of Aurora-A was 8.96 in PG cells, 21.13 in A549 cells, and 6.43 in NCI-H460 cells, respectively. The proportion of cells with tetraploid DNA was 19.88% in PG cells, 14.97% in A549 cells, and 10.6% in NCI-H460 cells, respectively (P<0.01); the proportion of cells with polyploid DNA was 2.66% in PG cells, 3.59% in A549 cells, and 2.30% in NCI-H460 cells, respectively. CONCLUSION: Aurora-A is overexpressed in the 3 lung cancer cell lines, but the mRNA levels are different.

[Reversal of resistance to adriamycin in human breast cancer cell line MCF-7/ADM by beta-elemene].

OBJECTIVE: To study the reversal mechanism of adriamycin (ADM) resistance in human breast cancer cell line MCF-7/ADM by beta-elemene (beta-ELE), a wide spectrum anticancer drug derived from the Chinese herb Curcuma chaeocaulis. METHODS: Sensitivity to ADM of MCF-7/ADM cells was studied by MTT assay. Intracellular accumulation of ADM in MCF-7/ADM cells was observed by fluorescent-spectrophotometry. Expression of bcl-2 protein was detected by flow cytometry. RESULTS: A non-cytotoxic dose (6 micro g/ml) and a weakly cytotoxic dose (13 micro g/ml) of beta-ELE could significantly enhance the cytotoxic effects of ADM on MCF-7/ADM cells to 1.4 and 2.2 fold as compared to the beta-ELE untreated control cells. The intracellular concentration of ADM in MCF-7/ADM cells was significantly increased after treatment with beta-ELE (P < 0.01). The expression of bcl-2 protein in MCF-7/ADM cells was reduced from 90.2% to 70.0% (P < 0.05). CONCLUSION: beta-ELE could partially reverse the drug resistance to ADM in MCF-7/ADM, which is related to the increased accumulation of intracellular ADM and the decreased expression of bcl-2.

[Abnormalities of molecular biology in premalignant lung lesions].

In recent twenty years, the incidence of lung cancer has increased quickly in our country. Till now, the morbidity and mortality of lung cancer have occupied the top of the cancers. Early diagnosis is the most important key to increase the five-year survival rate. This review is attempted to summarize the early hereditary events during the development of lung cancer, mainly including the mutation of p53 gene, the abnormal methylation of the promoter area of p16 gene, the loss of heterozygosity of 3p, 8p, 9p, 5q, etc. The purpose is to seek the biological markers during the development of lung cancer to provide theoretical basis for treatment of lung cancer.

[Effect of tanshinone microemulsion on reversing MDR in human tumor cells].

OBJECTIVE: To study the effect of tanshinone microemulsion (Tan-M) on the cytotoxicity to human leukemia-cell-line (K562/ADM) and the reversion of MDR in vitro. METHOD: Microemulsion being supposed as the control group, MT method is adopted to test cytotoxicity and the reverse of MDR. RESULT: Obvious cytotoxicity to K562/ADM was observed for tan-M. Cell non-toxic dosage (growth quotiety > 95%) of Tan-M is 0.2 microg x mL(-1). Low toxic dosage (growth quotiety 85-90%) was 0.7 microg x mL(-1). Cell non-toxic dosage of was 0.7 microg x mL(-1) and low toxic dosage was 1.2 microg x mL(-1). Cell non-toxic dosage of Tan-M (0.2 microg x mL(-1)) significantly lowered the IC50 of K562/ADM by ADM (P < 0.01), and reversed MDR was 3.88 times. Low toxic dosage of Tan-M reversed MDR was 3.97 times. E-M (0.2 microg x mL(-1)) reversed MDR was 2.62 times. CONCLUSION: The result indicates that tanshinone microemulsion possesses cell-toxic effects on human leukemia cell-line and may reverse MDR of tumor cells.

Effects of PKC activation on the meiotic maturation, fertilization and early embryonic development of mouse oocytes.

Protein kinase C (PKC) is a family of Ser/Thr protein kinase widely distributed in eukaryotes. There is evidence that PKC plays key roles in the meiotic maturation and activation of mammalian oocytes. However, the mechanism of PKC's actions and the PKC isoforms responsible for these actions are poorly understood. In this study, we reveal in mouse eggs and early embryos: (1) the effects of PKC on the meiotic and mitotic cell cycle progression during oocyte maturation, egg activation and embryonic cleavages; (2) the functional importance of classical PKC subclasses in these processes; and (3) the subcellular localization of the PKC alpha isoform during development from GV stage oocytes to the blastocyst stage embryos. The results indicate that the PKC activator phorbol 12-myristate 13-acetate (PMA) inhibits the meiotic resumption of cumulus-free mouse oocytes by a mechanism dependent not only on classical PKC activity but also on other PKC isoforms. PKC activation after germinal vesicle breakdown leads to the inhibition of mitogen-activated protein kinase phosphorylation and the arrest of cell cycle at MI stage. The second polar body emission and the cleavages of early embryos are blocked after prolonged PKC activation. The subcellular localization of PKC alpha isoform in mouse oocytes and embryos is developmental-stage associated. All these results suggest that PKC has multiple functional roles in the cell cycle progression of mouse oocytes and embryos.

Arsenic trioxide in the mechanism of drug resistance reversal in MCF-7/ADM cell line of human breast cancer.

OBJECTIVE: To investigate the effect of drug resistance by arsenic trioxide (As(2)O(3)) and its possible mechanism in human breast cancer cell line MCF-7/ADM. METHODS: Cytotoxicity of As(2)O(3) and the sensibility to adriamycin (ADM) in MCF-7/ADM cell line, a ADM-resistance cell line of human breast cancer, were studied through MTT assay. The concentration of intracellular ADM was detected by spectrofluorometry. With MCF-7/ADM cells treated with As(2)O(3) in combination with ADM, the glutathione-s-transferase (GST) activity was measured by biochemical method. The expression of GST-pi mRNA was assessed by RT-PCR. RESULTS: The non-cytotoxic dose of As(2)O(3) was 0.2 micro mol/L and the low cytotoxic dose was 0.8 micro mol/L to MCF-7/ADM cell line. 0.2 micro mol/L As(2)O(3) could significantly increase the intracellular accumulation of ADM in MCF-7/ADM cell line (P < 0.05). The medium inhibition concentration (IC(50)) was obviously reduced from 53.74 micro mol/L to 25.0 micro mol/L, with a reversal ratio of 2.1 as compared to its parental cell line. Before and after 0.2 micro mol/L, 0.8 micro mol/L As(2)O(3) were given, GST activities were decreased from 29.68 +/- 0.29 U/ml to 19.29 +/- 2.10 U/m l and 12.66 +/- 2.78 U/ml (P < 0.05). In addition, MCF-7/ADM cell line had overexpression of GST-pi mRNA. A significant down regulation of GST-pi mRNA was observed in MCF-7/ADM cells when As(2)O(3) and ADM (21.55 micro mol/L) were given for 24 hours. CONCLUSION: As(2)O(3) is able to enhance the cytotoxicity of ADM and partly reverse the ADM resistance of MCF-7/ADM cell line of human breast cancer, which may be related to the variation of GST-pi enzyme.

[Fine deletion mapping on chromosome 8p21 - 8p22 in adenocarcinoma of lung].

OBJECTIVE: To determine the common deletion region of loss of heterozygosity (LOH) on chromosome 8p21-8p22 in adenocarcinomas of lung, and facilitate identification of candidate tumor suppressor genes associated with adenocarcinoma of lung. METHODS: PCR and microsatellite analysis were used to examine the LOH frequency of 17 microsatellite loci at the 8p21-8p22 in the samples resected from 32 patients with lung adenocarcinoma and adenosquamous carcinoma. The relationship of LOH for each marker to pathological grade and that to clinical stage are investigated. RESULTS: Thirty-one out of the 32 (96.67%) samples showed allelic loss in at least one of the 17 markers. The most frequent LOH loci were mainly located in the three regions: D8S254-261, D8S1827-1731, and D8S1135 loci. Among them, the LOH frequency of D8S261 locus was related to the stage of tumor (P < 0.05). CONCLUSION: An interval of common deletion on chromosome 8p22, encompassing D8S254-261, D8S1827-1731 and D8S1135, might harbor candidate tumor suppressor gene(s) associated with pathogenesis of adenocarcinoma of lung.