Transcriptomics Reveals the ERF2-bHLH2-CML5 Module Responses to H2S and ROS in Postharvest Calcium Deficiency Apples.

Calcium deficiency usually causes accelerated quality deterioration in postharvest fruit, whereas the underlining mechanism is still unclear. Here, we report that calcium deficiency induced the development of bitter pit on the surface of apple peels compared with the healthy appearance in control apples during postharvest storage. Physiological analysis indicates that calcium-deficient peels contained higher levels of superoxide anion (O2•-), malondialdehyde (MDA), total phenol, flavonoid contents and polyphenol oxidase (PPO) activity, and reduced calcium, H2S production, anthocyanin, soluble protein content, and peroxidase (POD) activity compared with those in calcium-sufficient peels. The principal component analysis (PCA) results show that calcium content, ROS, and H2S production were the main factors between calcium-deficient and calcium-sufficient apple peels. Transcriptome data indicated that four calmodulin-like proteins (CMLs), seven AP2/ERFs, and three bHLHs transcripts were significantly differentially expressed in calcium-deficient apple peels. RT-qPCR and correlation analyses further revealed that CML5 expression was significantly positively correlated with the expression of ERF2/17, bHLH2, and H2S production related genes. In addition, transcriptional co-activation of CML5 by ERF2 and bHLH2 was demonstrated by apple transient expression assays and dual-luciferase reporter system experiments. Therefore, these findings provide a basis for studying the molecular mechanism of postharvest quality decline in calcium-deficient apples and the potential interaction between Ca2+ and endogenous H2S.

Storage Property Is Positively Correlated With Antioxidant Capacity in Different Sweet Potato Cultivars.

Sweet potato decays easily due to its high respiration rate and reactive oxygen species (ROS) accumulation during postharvest storage. In this study, we explored the relationship between antioxidant capacity in leaves and storage properties in different sweet potato cultivars, the tuberous roots of 10 sweet potato cultivars were used as the experimental materials to analyze the storage property during storage at 11-15°C. According to the decay percentage after 290 days of storage, Xu 32 was defined as a storage-tolerant cultivar (rot percentage less than 25%); Xu 55-2, Z 15-1, Shangshu 19, Yushu, and Zhezi 3 as above-moderate storage-tolerant cultivars (rot percentage ranging from 25 to 50%); Sushu 16, Yanshu 5, and Hanzi as medium-storable cultivars (rot percentage 50-75%); and Yan 25 as a storage-sensitive cultivar (rot percentage greater than 75%). Meanwhile, analysis of the α-amylase activity in root tubers of the 10 sweet potato cultivars during storage indicated that α-amylase activity was lowest in the storage-tolerant cultivar Xu 32 and highest in the storage-sensitive cultivar Yan 25. Evaluation of antioxidant enzyme activities and ROS content in the leaves of these 10 cultivars demonstrated that cultivar Xu 32, which showed the best storage property, had higher antioxidant enzyme activity [superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and peroxidase (POD)] but lower lipoxygenase (LOX) activity, hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents, and superoxide anion radical (O2⋅-) production rates compared with those of the storage-sensitive cultivar Yan 25 and the medium-storability cultivars Hanzi, Yanshu 5, and Sushu 16. Additionally, principal component analysis (PCA) suggested that sweet potato cultivars with different storage properties were clustered separately. Correlation and heat map analysis further indicated that CAT, APX, POD, and SOD activities were negatively correlated with α-amylase activity, while LOX activity and MDA and H2O2 contents were negatively correlated with the storage property of sweet potato. Combined, our findings revealed that storage property is highly correlated with antioxidant capacity in sweet potato leaves and negatively correlated with α-amylase activity in tuberous roots, which provides a convenient means for the screening of storage-tolerant sweet potato cultivars.

[Efficacy Analysis of Unrelated Umbilical Cord Blood Transplantation for the Treatment of Wiskott-Aldrich Syndrome].

OBJECTIVE: To explore the clinical efficacy and safety of unrelated umbilical cord blood transplantation (UCBT) for the treatment of Wiskott-Aldrich syndrome(WAS). METHODS: Five pediatric patients with WAS received single UCBT were retrospectively analyzed. The median age of these male patients was 268 days (range, 3 days -695 days). Among them, 2 patients were transplanted with a 6/6 matched cord blood graft，the other 3 patients received a 5/6 matched cord blood graft. Myeloablative conditioning regimen was applied, and all patients received a combination of cyclosporine and mycophenolate mofetil for the prophylaxis of graft versus host disease (GVHD). The recovery time of neutrophils and platelets as well as chimerism after transplantation were taken as the evidence of hematopoietic reconstruction. RESULTS: All the five pediatric patients had hematopoietic recovery. A median time of neutrophil cells after transplantation was at 15.8 days (range,11 days -25 days), and platelet recovery was at a median of 20.4 days(range,12 days-30 days). Chimerism data were available for 5 patients at 30 days after UCBT, 4 out of the 5 patients had full donor chimerism and only one patient had mixed chimerism. There were 2 cases with pre-engraftment syndrome, 3 cases with acute GVHD gradeⅠ-Ⅲ, 4 cases with pulmonary infection and cytomegalovirus infection, but chronic GVHD was not observed in 5 cases. Four patients were alive with a median follow-up of 12.3 months (range, 5 months-17 months), and one patient had died at 22 days after UCBT. CONCLUSION: Unrelated umbilical cord blood transplantation is a safe and effective treatment method for Wiskott-Aldrich syndrome.

[Effect of gambogic acid on proliferation of SKM-1 cells and its mechanism].

The aim of this study was to explore the effect of gambogic acid (GA) on MDS SKM-1 cell proliferation, apoptosis and their possible mechanism. Cell proliferation was determined by MTT method. The apoptosis percentage and cell cycle regulation of SKM-1 cells were analyzed by flow cytometry. Morphological features were observed by light microscopy. The mRNA expression of bcl-2 and bax were detected by RT-PCR. The results showed that GA could inhibit the proliferation of SKM-1 cells in a dose- and time-dependent manner (IC50 was 0.37 µg/ml at 48 h), increase the apoptotic percentage of SKM-1 cells, and arrest cell cycle at the G0/G1. The expression of bax mRNA was up-regulated while that of bcl-2 mRNA was down-regulated in SKM-1 cells treated with GA for 48 h. It is concluded that GA can induce apoptosis, which may be related to its effect of arresting cells at phase of G0/G1 and down-regulating bcl-2/bax ratio.

[Effect of desferrioxamine on K562/A02 cell line and its mechanism].

Iron is an essential element for cell growing including tumor cells. This study was purposed to explore the effect of desferrioxamine (DFO) on cell line K562/A02 and its mechanism. K562/A02 cells were cultured with different concentrations of DFO. The inhibitory effects of adriamycin (ADM) used alone or combined with DFO on the proliferation of K562/A02 was evaluated by MTT assay. The apoptosis rate of K562/A02 cells after treatment with 0, 12.5, 25 and 50 µmol/L DFO alone or in combination with 1 mg/L ADM were analyzed by flow cytometry. ADM accumulation in K562/A02 cells after treatment with different concentrations of 0, 12.5, 25 and 50 µmol/L DFO were also analyzed by flow cytometry. The levels of BAX/BCL-2 and MDR1 mRNA were determined by RT-PCR, and then the protein level of P-glycoprotein (P-gp) was detected by Western blot. The results showed that the IC(50) of ADM for K562 and K562/A02 cells were (1.46 ± 0.07) mg/L and (40.98 ± 3.05) mg/L respectively. The resistance of K562/A02 cells to ADM was 28.06 times as that of K562 cells. After treatment of K562/A02 cell with DFO of 12.5, 25 and 50 µmol/L for 48 hours, the resistance of K562/A02 cells to ADM were increased by 24.95, 16.11 and 9.99 times respectively. When K562/A02 cells were incubated with different concentrations of DFO of 12.5, 25, 50 µmol/L for 48 hours, the apoptosis rat were (3.50 ± 0.30)%, (7.27 ± 0.32)% and (12.53 ± 1.21)% respectively. After co-culture with DFO and ADM for 48 hours, apoptosis rate were (6.13 ± 0.29)%, (9.57 ± 0.40)% and (18.97 ± 1.10)% respectively. The above apoptosis rates was much higher than that of control group (p < 0.05) and they were dose-dependent. In comparison between DFO + ADM group and DFO group, there was no significant difference (p > 0.05). Expression rate of BAX/BCL-2 increased. The levels of MDR1 mRNA reduced. Furthermore, expression of P-gp also decreased in K562/A02 cells. It is concluded that iron increase can promote K562/A02 cells growth and inhibit their apoptosis. Otherwise, iron-deprivation can induce K562/A02 cells apoptosis. DFO disturbs the iron metabolism and inhibits DNA synthesis of K562/A02 cells. This action of DFO may enhance the susceptibility of K562/A02 cells to apoptosis induced by chemotherapeutic drugs. The iron-deprivation may play a role in the treatment of leukemia with combination of DFO with other anticancer agents.

[Detection of bcr-abl fusion gene mRNA level in K562/A02 cell line by real-time quantitative RT-PCR].

This study was aimed to quantitatively analyze the mRNA level of bcr-abl fusion gene in K562/A02 cell line by real-time quantitative reverse transcriptase polymerase chain reaction (RQ-RT-PCR) technique. After being cultured for a period of time, the K562/A02 cell line was collected and RNA was extracted using TRIzoL kit. The real-time quantitative reverse transcriptase polymerase chain reaction technology was used to detect the level of bcr-abl fusion gene and internal reference abl gene. The results showed that a fine reproducibility was obtained between 10(7) and 10(3) copies/ml, reproducible sensitivity of RQ-RT-PCR was 10(-5). The expression of bcr-abl fusion gene in K562/A02 cells was higher and the level of bcr-abl mRNA was more than 100% in K562/A02 cells. It is concluded that RQ-RT-PCR is a reliable, sensitive and reproducible method for detecting mRNA level of bcr-abl fusion gene, which may be useful in monitoring the chronic myeloid leukemia.

[Effect of hypoxia inducible factor1-α inhibitor on reversal of multidrug resistance of K562/A02 cell line].

OBJECTIVE: To study the reversal effect of the hypoxia inducible factor (HIF)-1α inhibitor, YC-1, on multidrug resistance of K562/A02 cells and its mechanism. METHODS: Pre- and post- incubation with adriamycin (ADM) alone or in combination with YC-1 for 48 h, the proliferation capacity of K562/A02 and K562 cells were evaluated by MTT assay. The apoptosis rate of K562/A02 cells after treated with 0, 5, 10 and 20 µmol/L YC-1 alone or in combination with 1 mg/L ADM and intracellular ADM concentration were analyzed by flow cytometry (FCM). The mRNA levels of HIF-1α and mdr1 genes were determined by semi-quantitative RT-PCR. The protein levels of HIF-1α and P-glycoprotein (P-gp) were detected by Western blot. RESULTS: The IC(50) of ADM for K562 and K562/A02 cells were (1.56 ± 0.07) mg/L and (42.98 ± 3.15) mg/L respectively. The resistance of K562/A02 cells to ADM was 27.55- fold higher of that of K562 cells. After treatment with YC-1 (5 µmol/L, 10 µmol/L, 20 µmol/L) for 48h, the resistances of K562/A02 cells to ADM were 24.63-, 16.38- and 10.71- fold increase respectively. After treatment of K562/A02 cell with YC-1 (0 µmol/L, 5 µmol/L, 10 µmol/L, 20 µmol/L) alone or in combination with 1 mg/L ADM for 48 h, the apoptotic rates were (1.9 ± 0.9)%, (4.9 ± 0.9)%, (5.8 ± 1.1)%, and (9.3 ± 1.4)% and (2.3 ± 0.7)%, (8.2 ± 1.2)%, (19.0 ± 1.7)%, and (34.5 ± 2.4)% respectively. The intracellular flucorescence intensity of ADM were 232 ± 33, 1300 ± 219, 1961 ± 240 and 3342 ± 269 in the combined treatment group. With the increase in YC-1 concentration, the levels of mdr1 mRNA reduced, while that of HIF-1α mRNA had no obvious change. Furthermore, the expressions of HIF-1α and P-gp were also decreased in K562/A02 cells. CONCLUSION: YC-1, as a HIF-1 inhibitor, can reverse multidrug resistance of K562/A02 cells through down-regulating HIF-1α and P-gp.

[Effect of sodium valproate on human myelodysplastic syndrome cell line SKM-1 and its mechanism].

This study was aimed to investigate the effect of sodium valproate(VPA) on human myelodysplastic syndrome cell line SKM-1 and its mechanism. The cell proliferation was determined by MTT assay, cell apoptosis was analyzed by flow cytometry. The expressions of c-flipl, c-flips and dlk1 mRNA were detected by RT-PCR. The results showed that VPA could inhibited the growth of SKM-1 cells in dose- and time-dependent manners. The flow cytometric analysis indicated that VPA could induce cell apoptosis, apoptosis rate increased in dose-dependent manner. The expressions of c-flipl, c-flips and dlk1 mRNA in SKM-1 cell treated with VPA decreased using of VPA. It is concluded that VPA can induce apoptosis and inhibited proliferation of SKM-1 cells. In this process, the decreasing of c-flipl, c-flips and dlk1 mRNA expression may play important roles.

[Gene expression in patients with myelodysplastic syndrome].

This study was aimed to investigate the expression of c-FLIPL, c-FLIPS and DLK1 mRNA in the patients with myelodysplastic syndrome (MDS) and its clinical significance. The mRNA expression of c-FLIPL, c-FLIPS and DLK1 in bone marrow mononuclear cells (BMMNC) of 16 patients with MDS and 3 controls were detected by RT-PCR. The results indicated that the expression of DLK1 mRNA was up-regulated in MDS, including RA and RAEB, as compared with controls (p < 0.05). There was no significant difference in expression of DLK1 between RA and RAEB patients (p > 0.05); the expression of c-FLIPL mRNA both in RA and RAEB patients was higher than that in controls (p < 0.05). There was no significant difference in expression of c-FLIPL between RA and RAEB patients (p > 0.05); the expression of c-FLIPS mRNA was not significantly different between MDS patients and controls (p > 0.05), but its expression in RAEB patients was significantly higher as compared with RA patients and controls (p < 0.05). It is concluded that the mRNA expressions of DLK1, c-FLIPL and c-FLIPS in MDS patients are abnormal, some of which may be useful as an important indicator for the evaluation of development in MDS.

[Effects of hypoxia-inducible factor inhibitor on expression of HIF-1alpha and VEGF and induction of apoptosis in leukemic cell lines].

This study was purposed to investigate the effect of a hypoxia-inducible factor inhibitor (YC-1) on expression of hypoxia-inducible factor 1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) as well as induction of apoptosis in leukemic cell lines. RT-PCR was used to determine the levels of HIF-1alpha mRNA and VEGF mRNA in K562, U937 and Jurkat cells. After treatment of U937 cell with 4 micromol/L YC-1, cell apoptosis was assayed by DAPI staining under fluorescent microscope and flow cytometry with Annexin V-FITC/PI staining; the expression levels of HIF-1alpha mRNA and VEGF mRNA were measured with RT-PCR; the expression levels of HIF-1alpha, VEGF, BAX, BCL-2 and caspase-3 proteins were measured by Western blot. The results showed that HIF-1alpha mRNA and VEGF mRNA were expressed in all three leukemia cell lines. After treatment of U937 cell with 4 micromol/L YC-1 for 0, 8, 16 and 24 hours, the changes of morphologic features of U937 cells could be observed under fluorescent microscope and the apoptotic rates significantly increased in time-dependent manner, they were (4.87 +/- 0.70)%, (27.27 +/- 2.00)%, (51.53 +/- 2.81) and (60.5 +/- 3.20)% respectively, the expression levels of VEGF mRNA reduced, while the expression levels of HIF-1alpha mRNA had no obviously changes.Furthermore, the expression of HIF-1alpha, VEGF and BCL-2 decreased, while the expression of BAX and caspase-3 increased, the ratio of BAX/BCL-2 increased in time-dependent manner (r = 0.973, p < 0.01). It is concluded that HIF-1alpha mRNA and VEGF mRNA are all expressed in in K562, U937 and Jurkat cells, YC-1 has significant effect on down-regulating the protein expression of HIF-1alpha and VEGF, and induces the apoptosis in U937. The mechanism of apoptosis in leukemic cells may involve in up-regulating BAX/BCL-2 ratio and expression of protein caspase-3.

[Effect of PDMP, a glucosylceramide synthase inhibitor, on reversion of daunorubicin resistance in human leukemia cell line K562/A02].

This study was purposed to investigate the reversal effect of glucosylceramide synthase (GCS) inhibitor D, L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) hydrochloride, on multidrug resistance in K562/A02 cells and its mechanism. The IC(50) (the half maximal inhibitory concentration) of PDMP was measured by MTT method. Cell apoptosis and intracellular daunorubicin (DNR) concentration were detected by flow cytometry. The expression of GCS and mdr1 genes were assayed by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot. The results showed that the IC(50) of DNR in K562 and K562/A02 cells were 0.23 +/- 0.02 and 7.15 +/- 0.24 microg/ml respectively. When the concentration of PDMP was equal to or less than 20 micromol/L ( < / = 20 micromol/L), the obviously inhibitory effect on proliferation of K562 and K562/A02 cells was not observed, but both 20 micromol/L and 10 micromol/L PDMP could enhance the sensitivity of K562/A02 cells to DNR (p < 0.01) and the reversal multiples were 2.59 and 1.69 respectively. After treating with 20 micromol/L and 10 micromol/L PDMP for 48 hours, the concentration of DNR in K562/A02 cells increased (p < 0.05) and the apoptotic rate also was elevated (p < 0.01). The expressions of GCS and mdr1 genes were down-regulated at mRNA and protein levels after treating K562/A02 cells with 20 micromol/L PDMP for 48 hours. It is concluded that PDMP can enhance the sensitivity of K562/A02 cells to DNR by increasing cell apoptosis rate and accumulation concentration of DNR in cells, which may be related to down-regulated expressions of GCS and mdr1 genes.

[Reversal of multidrug-resistance in human leukemia cell line K562/A02 by tyrosine kinase inhibitors].

This study was aimed to investigate the reversal effect of tyrosine kinase inhibitors (TKI) Imatinib and Nilotinib on multidrug-resistant cell line K562/A02. The expression levels of mdr-1 mRNA and bcr-abl mRNA were assayed by RT-PCR. The protein levels of P-glycoprotein (P-gp) and P210 were detected by Western blot. The daunorubicin (DNR) accumulation in K562/A02 cells were analyzed by flow cytometry (FCM). The results showed that the 0.0625 micromol/L Imatinib or 5 nmol/L Nilotinib alone had no cytotoxic effect on the inhibition of K562/A02 cells. When K562/A02 cells were treated with Imatinib or Nilotinib alone for 48 hours, the expressions of mdr-1 mRNA, der/abl mRNA, P-gp and P210 protein were all down-regulated, furthermore the effect of Nilotinib was stronger than that of Imatinib. The detection of fluorescence intensity revealed that the DNR concentration in K562/A02 cells treated with Imatinib or Nilotinib alone for 48 hours were 7.85% and 12.02% of K562 cells respectively. It is concluded that the tyrosine kinase inhibitors show great effect reversing drug resistance of cells, moreover, the effect of Nilotinib is stronger than that of Imatinib.

[Construction and identification of Mdr-1-shRNA eukaryotic expression vector].

This study was purposed to construct and identify the short hairpin RNA (shRNA) eukaryotic expression vector for targeting gene mdr-1 which may play an important role in K562/A02. Short hairpin RNA (shRNA) aiming at the target sequence was to synthesized, the 3491-3509, 1539-1557and 3103-3121 nucleotide of mdr-1 mRNA were selected as targets. The selected nucleotides were cloned in the plasmid pGCSilencer-U6-neo-GFP respectively, and the resultant recombinant plasmids were named as pGY1-1, pGY1-2 and pGY1-3. The sequences of the recombinant plasmids were identified by DNA sequencing and PCR electrophoresis. The recombinant plasmids were transfected into the cell line K562/A02 by lipofection. After being transfected for 48 hours, the inhibition of mdr-1 mRNA was detected by real time-PCR, and P-gp expression was detected by Western blot. The results showed that the specific oligonucleotide was cloned into the vector successfully, and the expression of mdr-1 mRNA and P-gp in K562/A02 cells was reduced after transfecting the recombinant plasmid, as compared to the control group. It is concluded that the shRNA eukaryotic expression vector has been successfully established which can inhibit the expression of mdr-1 mRNA, setting up the basis to futher explore the effects of mdr-1 on cell line of K562/A02.

[Analysis on laboratory and clinical characteristics in 65 cases of myelodysplastic syndrome].

The aim of this study was to gain more insight into the understanding of myelodysplastic syndrome in the clinical and laboratory features. The clinical data of 65 patients with MDS were reviewed and analysed. According to FAB criteria, 65 patients were classified as follows: 27 patients with RA, 1 patient with RAS, 33 patients with RAEB, 3 patients with RAEB-T, and 1 patient with CMML. The median age of them was 66 years old (range 19-89 years), and 6 patients had a history of toxic exposure (secondary MDS). The bone marrow smears, bone marrow biopsy and cytogenetic examinations were performed in this study. The results showed that dysplasia was found in 64 patients examined with bone marrow smears (98.5%), among them trilineage dysplasia in 21 patients (32.3%), bilineage dysplasia in 33 patients (50.8%), only erythroid dysplasia in 8 cases (12.3%) and 2 patients (3.1%) only with myeloid dysplasia. The bone marrow biopsy was performed in 38 patients, abnormal localization of immature precursor (ALIP) occurred in 6 cases. 29 patients had abnormal karyotypes, accounting for 59.2% of the 49 patients subjected cytogenetic examination. The abnormal chromosome was the major cytogenetic abnormality, which occurred more often in secondary MDS and the patients with RAEB or RAEB-T. Among the 49 patients who had received cytogenetic examination, 15 patients transformed into AML with the incidence of 30.61%, but only 3 out of 20 patients in the group of normal chromosome transformed into AML (15%), while 12 out of 29 patients in the group of abnormal karyotypes transformed into AML (41.4%). The median time of following up was 35 months (range 2 - 106 months). The median survival time was 26.8 months and 8 months in the patients with normal karyotype and chromosome aberrations respectively. In conclusion, the incidence of MDS in our country is younger than that in Western countries, the rate of abnormal chromosome in high risk MDS is higher than that in low risk MDS. Meanwhile, those who have the change of chromosome are related to the transformation of MDS into AML and have shorter survival time than those MDS patients with normal karyotypes.

[Auto-hematopoietic stem cell transplantation for multiple myeloma accompanied with amyloidosis in four limbs].

The study was aimed to investigate the effective therapeutic method for patients with multiple myeloma accompanied with amyloidosis. A 58-year-old patient diagnosed as multiple myeloma accompanied with amyloidosis in four limbs was enrolled in this study. The various clinical and laboratorial examinations were performed, including bone marrow smear, immunologic test, radiography and so on. Patient received chemotherapeutic drugs and then autologous hematopoietic stem cell transplantation (auto-HSCT). The result showed that hematopoietic reconstitution was achieved at 23 days after auto-HSCT. Immunofixation electrophoresis was normal. There was only 0.6% plasma cells in the bone marrow. In conclusion, the auto-HSCT may be an effective treatment for multiple myeloma accompanied with amyloidosis in four limbs.

[Inhibition effect of gambogic acid on MUTZ-1 cell line and its possible mechanism].

This study was aimed to investigate the effects of gambogic acid on the cells of high-risk patients with myelodysplastic syndrome (MDS) in vitro and its mechanism. The inhibition effect of gambogic acid on growth of MUTZ-1 cell line of MDS-RAEB was detected by MTT method. Apoptosis and cell cycle were detected by morphological observation and flow cytometry respectively. The expressions of bax/bcl-2 gene at mRNA level were detected by RT-PCR. The results indicated that the Gambogic acid inhibited the growth of MUTZ-1 cells, the inhibitory rate of gambogic acid with the range of 0.2 - 0.8 microg/ml was enhanced along with increasing of drug concentration. Flow cytometric assay showed that the apoptotic rate of MUTZ-1 cells treated by gambogic acid also was enhanced along with increasing of drug concentration, the apoptotic rates resulting from gambogic acid (0, 0.4, 0.6, 0.8 microg/ml) were (5 +/- 0.5)%, (13 +/- 0.5)%, (37 +/- 0.7)% and (56 +/- 0.6)% respectively. The characteristic changes of apoptosis emerged in MUTZ-1 cells after being exposed to gambogic acid. Gambogic acid could significantly down-regulate the expressions of bcl-2 gene in a dose dependent manner, however, it had no effects on bax gene. It is concluded that within the range of concentration from 0.4 to 0. 8 microg/ml, gambogic acid can inhibit the growth of MUTZ-1 cells by inducing their apoptosis and down-regulating the expression of bcl-2 gene, which may be one of the main mechanisms underlying its antitumor effects.

MDR reversal activity of bromotetrandrine in vitro and in vivo.

The present study was aimed to evaluate the MDR reversal activity of bromotetrandrine (BrTet) in vitro and in vivo. The inhibitory effects of adriamycin (ADM) used alone or in combination with BrTet or Tet on the proliferation of K562 and K562/A02 cells were evaluated by MTT assay. The ADM accumulation and the protein levels of P-glycoprotein (P-gp) were detected by flow cytometry. The mRNA levels of P-gp were determined by RT-PCR. The in vivo effect of BrTet and Tet was investigated by using nude mice grafted with sensitive human leukemia cell line K562 and MDR cell line K562/A02. The results showed that BrTet at 0.25, 0.5 and 1 micromol/L reversed the resistance to ADM in MDR K562/A02 cells in a dose-dependent manner. Flow cytometry suggested that BrTet significantly increased the intracellular accumulation of ADM in K562/A02 cells in a dose-dependent manner. BrTet also inhibited the overexpression of P-gp in K562/A02 cells, and down-regulated mdr1 expression. In nude mice bearing K562 xenografts on the left flank and K562/A02 xenografts on the right flank, intraperitoneal injection of 10 mg/kg BrTet significantly enhanced the antitumor activity of ADM against K562/A02 xenografts with inhibitory rates of 26.1%, while ADM alone inhibited the growth of K562/A02 xenografts only by 5.8%. No enhancement effect by BrTet was seen in K562 xenografts. It is concluded that BrTet shows significant MDR reversal activity in vitro and in vivo. Its activity may be related to the inhibition of P-gp overexpression and the increase intracellular accumulation of anticancer drugs. BrTet may be a promising-MDR modulator for eventual assessment in the clinic.

[Effect of tetrandrine, toremifene and their combination on the reversion of multidrug resistance of K562/A02 cell line].

This study was aimed to investigate the reversible effect of tetrandrine, toremifene and their combination on multidrug resistance of K562/A02 cell line. The IC(50) (the concentration causing 50% inhibition of cell growth) of adriamycin (ADR) were assayed by MTT method, the expression of MDR1 mRNA was measured by RT-PCR, the concentration of p-glycoprotein (P-gp) and intracellular ADR were detected by flow cytometry. The results showed that the IC(50) of ADR on K562/A02 and K562 cells were 57.43 and 1.16 mg/L, respectively. The IC(50) of ADR on K562/A02 cells after treatment with tetrandrine, toremifene and both combination were 14.12, 20.74 and 9.14 mg/L respectively, but both drugs did not influence the IC(50) of ADR on K562 cells. Pretreating K562/A02 cells with toremifene (2.5 micromol/L), tetrandrine (1 micromol/L) or both for 72 hours partially restored the sensitivity of K562/A02 cells to ADR. Tetrandrine and toremifene (alone or combination) elevated the ADR concentration in K562/A02, down regulated the expressions of P-gp and MDR1 mRNA. It is concluded that multidrug resistance of K562/A02 cells can be partially reversed by tetrandrine or toremifene, the combination of both drugs shows a higher synergistic reversal effect.

[Enhancement of reversing drug resistance of K562/A02 cells to adriamycin by ultrasound-induced cavitation].

This study was aimed to investigate the effects of low frequency and power ultrasound combined with adriamycin on apoptosis of drug-resistant leukemia cell line K562/A02 in vitro, to find out the parameters of optimal exposure, and to explore the possible mechanism reversing drug-resistance of K562/A02 cells. The K562/A02 cells in logarithmic growth phase were used in experiments. The experiments were divided into 4 groups: group control, group adriamycin (A02) alone, group ultrasound (US) alone and group A02+US. The trypan blue dye exclusion test and MTT assay were used to determine the cell viability; Wright's staining was used to detect the apoptosis; the flow cytometry was used to analyze the drug concentration, and the scanning electron microscopy was used to observe the changes of cell surface. The results showed that the significant differences in cell viability, intracellular adriamycin concentration and changes of cell membrane were found between ultrasound-treated and untreated cells in the presence of various concentration of adriamycin. The exposure to ultrasound at 20 kHZ, 0.25 W/cm2 for 60 seconds could obviously decrease LC50 of adriamycin to K562/A02 cells, while the exposure to ultrasound at 20 kHZ, 0.05 W/cm2 for 60 seconds could kill K562/A02 cells at once. After being treated by low frequency ultrasound, the small holes with diameter about 1-2 microm in the cell surface appeared. The ultrasound increased the adriamycin concentration in the cells, accelerated the formation of apoptotic bodies, and promoted apoptosis of adriamycin-resistant cells. It is concluded that the ultrasound at optimal parameters enhances inhibitory effect of adriamycin on drug-resistant cell line, thereby reverses drug-resistance of drug-resistant cell line through sound-hole effect in tumor cells resulting from ultrasound induced cavitation.

[Phenylhexyl isothiocyanate reducing U266 cell line methylation level of p16 gene].

This study was purposed to investigate whether phenylhexyl isothiocyanate (PHI) can reduce p16 gene methylation level or not. The myeloma U226 cell line was cultured with PHI of 0, 5, 10 micromol/L for 72 hours, then DNA was extracted. Hydrosulfite was used to treat the genome DNA of healthy adult, PCR amplification was carried out by using this DNA as template. The gene chip detecting methylation changes of 3 CpG in promoter region of p16 gene was constructed by designing a pair of probes which contain one methylated and one unmethylated probes. This pair of probes was used to detect 3 consecutive sites of CpG island in p16 gene. The standard curve was constructed by using gene chip after the methylated and unmethylated DNA were mixed at different ratio. Then treated samples of U266 cells were dotted on gene chip, obtained results were compared with standard curve to get the quantitative results. The results indicated that the probes on chip had excellent reproducible ability and precision, the methylation level of p16 gene in U266 cells treated with 0, 5 and 10 micromol/L of PHI was determined as 78.2%, 61.7% and 54.8%, respectively. It is concluded that the PHI can reduce the methylation level of p16 gene in U266 cells.