Liposomal amphotericin B in combination with caspofungin for invasive aspergillosis in patients with hematologic malignancies: a randomized pilot study (Combistrat trial)

BACKGROUND: Invasive aspergillosis (IA) has a poor prognosis in immunocompromised patients. Combinations of drugs that act on different targets are expected to improve the clinical efficacy of separate compounds. METHODS: Patients with proven or probable IA were randomized in a prospective, open pilot study to receive either a combination of liposomal amphotericin B (AmB) at the standard dose (3 mg/kg daily) and caspofungin at the standard dose or monotherapy with a high-dose AmB regimen (10 mg/kg daily). RESULTS: Thirty patients (21 men and 9 women) with hematologic malignancies were analyzed, and there were 15 patients in each arm. The median duration of treatment was 18 days for the combination group and 17 days for the high-dose monotherapy group. At the end of treatment, there were significantly more favorable overall responses (partial or complete responses; P = .028) in the combination group (10 of 15 patients; 67%) compared with the high-dose monotherapy group (4 of 15 patients; 27%). Survival rates at 12 weeks after inclusion were 100% and 80%, respectively. Infusion-related reactions occurred in 3 patients in the high-dose monotherapy group. A 2-fold increase in serum creatinine occurred in 4 of 17 patients (23%) who received high-dose monotherapy and 1 of 15 patient (7%) who received combination therapy; hypokalemia <3 mmol/L occurred in 3 patients and 2 patients, respectively. CONCLUSIONS: The combination of liposomal AmB and caspofungin was promising as therapy for IA compared with monotherapy. A trial that includes more patients will be required next to confirm the results of this pilot study.     

Antifungal activity of amphotericin B,caspofungin and posaconazole on Candida albicans biofilms in intermediate and mature development phases

The aim of this study was to examine the antifungal activity of amphotericin B, caspofungin and posaconazole on Candida albicans biofilms in the intermediate and mature development phases. Candida albicans biofilms, previously grown for either 24, 48 or 72 h in 96‐well microtitre plates, were treated for 48 h with amphotericin B, caspofungin or posaconazole in increasing concentrations according to the respective minimal inhibitory concentration (MIC) determined for planktonic cells (1–128 × MIC). The biofilms were quantified using the mean optical density (OD) determined by XTT assay. Antifungal activities were expressed as percentage of reduction in OD of drug‐treated biofilms compared to untreated biofilms. To test the fungicidal activity of antifungal agents, the unfixed biofilms were scraped off and seeded to Sabouraud agar. Caspofungin and amphotericin B showed higher activity against C. albicans biofilm grown for 24 h and 72 h (≥50% reduction of OD) than biofilms grown for 48 h, whereas posaconazole showed similar, but reduced activity against all phases of C. albicans biofilm (≤50% reduction of OD). Caspofungin at 1–4 × MIC achieved the greatest decrease in the biofilm OD grown for 24, 48 and 72 h, whereas amphotericin B showed dose‐dependent activity. However, all tested antifungals failed to reach fungicidal activity in all biofilm development phases.     

Multicentre surveillance study on feasibility,safety and efficacy of antifungal combination therapy for proven or probable invasive fungal diseases in haematological patients: the SEIFEM real‐life combo study

This multicentre observational study evaluated the feasibility, efficacy and toxicity of antifungal combination therapy (combo) as treatment of proven or probable invasive fungal diseases (IFDs) in patients with haematological malignancies. Between January 2005 and January 2010, 84 cases of IFDs (39 proven and 45 probable) treated with combo were collected in 20 Hematological Italian Centres, in patients who underwent chemotherapy or allogeneic haematopoietic stem cell transplantation for haematological diseases. Median age of patients was 34 years (range 1–73) and 37% had less than 18 years. Acute leukaemia was the most common underlying haematological disease (68/84; 81%). The phase of treatment was as follows: first induction in 21/84 (25%), consolidation phase in 18/84 (21%) and reinduction/salvage in 45/84 (54%). The main site of infection was lung with or without other sites. The principal fungal pathogens were as follows: Aspergillus sp. 68 cases (81%), Candida sp. six cases (8%), Zygomycetes four cases (5%) and Fusarium sp. four cases (5%). The most used combo was caspofungin+voriconazole 35/84 (42%), caspofungin + liposomal amphotericin B (L‐AmB) 20/84 (24%) and L‐AmB+voriconazole 15/84 (18%). The median duration of combo was 19 days (range 3–180). The overall response rate (ORR) was 73% (61/84 responders) without significant differences between the combo regimens. The most important factor that significantly influenced the response was granulocyte (PMN) recovery (P 0.009). Only one patient discontinued therapy (voriconazole‐related neurotoxicity) and 22% experienced mild and reversible adverse events (hypokalaemia, ALT/AST increase and creatinine increase). The IFDs‐attributable mortality was 17%. This study indicates that combo was both well tolerated and effective in haematological patients. The most used combo regimens were caspofungin + voriconazole (ORR 80%) and caspofungin + L‐AmB (ORR 70%). The ORR was 73% and the mortality IFD related was 17%. PMN recovery during combo predicts a favourable outcome. Clinical Trials Registration: NCT00906633.     

Antifungal susceptibility profile in vitro of Sporothrix schenckii in two growth phases and by two methods: microdilution and E‐test

The susceptibility profile of 91 Sporothrix schenckii isolates in both growth phases was determined by microdilution test (Antifungal Susceptibility Testing of the European Committee for Antimicrobial Susceptibility Testing; AFST‐EUCAST). Amphotericin B (AMB), itraconazole (ITC), posaconazole, ravuconazole and terbinafine were found active in vitro against both phases but minimum inhibitory concentrations values for mycelial phase were significantly higher. Fluconazole (FLC) and voriconazole (VRC) were inactive in vitro against both phases. The E‐test technique was also performed with 41 representative isolates for AMB, FLC, ITC and VRC. Average agreement rates between yeast phase microdilution results and E‐test results were high for AMB (77.5%) and FLC (87.8%), but low for ITC and VRC with rates of 56.4% and 54.5%, respectively. AFST‐EUCAST is not the most recommended test to perform drug susceptibility testing of S. schenckii in clinical laboratories, and E‐test could be an alternative methodology for this purpose, mainly when the activity in vitro of antifungal agents of AMB and FLC are evaluated.     

Stability in the cumulative incidence,severity and mortality of 101 cases of invasive mucormycosis in high‐risk patients from 1995 to 2011: a comparison of eras immediately before and after the availability of voriconazole and echinocandin‐amphotericin combination therapies

As invasive mucormycosis (IM) numbers rise, clinicians suspect prior voriconazole worsens IM incidence and severity, and believe combination anti‐fungal therapy improves IM survival. To compare the cumulative incidence (CI), severity and mortality of IM in eras immediately before and after the commercial availability of voriconazole all IM cases from 1995 to 2011 were analysed across four risk‐groups (hematologic/oncologic malignancy (H/O), stem cell transplantation (SCT), solid organ transplantation (SOT) and other), and two eras, E1 (1995–2003) and E2, (2004–2011). Of 101 IM cases, (79 proven, 22 probable): 30 were in E1 (3.3/year) and 71 in E2 (8.9/year). Between eras, the proportion with H/O or SCT rose from 47% to 73%, while ‘other’ dropped from 33% to 11% (P = 0.036). Between eras, the CI of IM did not significantly increase in SCT (P = 0.27) or SOT (P = 0.30), and patterns of anatomic location (P = 0.122) and surgical debridement (P = 0.200) were similar. Significantly more patients received amphotericin‐echinocandin combination therapy in E2 (31% vs. 5%, P = 0.01); however, 90‐day survival did not improve (54% vs. 59%, P = 0.67). Since 2003, the rise of IM reflects increasing numbers at risk, not prior use of voriconazole. Frequent combination of anti‐fungal therapy has not improved survival.     

Incidence and Management of Proven and Probable Fungal Infections in Patients with Acute Leukemia: A Single Center Experience

Abstract

The incidence of fungal infections and the role of liposomal amphotericin B (Ambisome) in proven and probable infections were evaluated in acute leukemic patients, intolerant to conventional amphotericin B. During 1999-2002, 307 febrile episodes occurred in 231 patients. Fungi were responsible for 3% of bloodstream infections. Ambisome was employed in 5 fungal sepsis (1 Candida albicans, 1 C. famata, 1 C. tropicalis, 1 C. krusei, 1 Geotrichum capitatum) 2 Aspergillosis, 2 probable fungal pneumonia cases. A favorable response was achieved in 78% of patients (4 fungemia, 2 aspergillosis, 1 probable), an unfavorable response in 1 C. krusei fungemia and in 1 probable pneumonia. Our antimicrobial pattern docu-mented a high resistance rate to azoles. We concluded that Ambisome is an effec-tive and well tolerated agent and its introduction has changed the outcome for many patients, although in some refractory diseases other strategies must be con-sidered.     

Current scenario of cryptococcosis and antifungal susceptibility pattern in India: a cause for reappraisal

This study analysed the spectrum, antifungal susceptibility pattern, clinical course and molecular epidemiology of cryptococcosis. Four hundred and thirty-nine samples obtained from 378 meningitis patients were processed by standard procedures. Minimum inhibitory concentration (MIC) of fluconazole and amphotericin B for the isolates was tested by broth micro dilution and by E-strip method. Molecular analysis by random amplified polymorphic DNA-PCR of eight isolates was performed using M13 primer. Cryptococcosis was diagnosed in 35 patients [HIV-1 seropositive (19) and apparently immunocompetent (16)]. Cryptococcus neoformans var. neoformans (serotype A and D) was the predominant isolate on phenotypic identification. Three C. neoformans var. gattii were isolated from HIV-1 seropositive (2) and apparently immunocompetent (1) patients. MIC 90 for amphotericin B and fluconazole were 1 and 8 μg ml⁻¹ respectively. On RAPD-PCR, less diversity was seen among Indian isolates. AIDS remains the single most important risk factor for cryptococcosis. Rising MIC of the available induction and maintenance drugs is of grave concern. The DNA typing technique showed less diversity among Indian strains. Routine surveillance and application of molecular typing methods are crucial to know the baseline and existing pattern of cryptococcosis.     

In vitro and in vivo antitumor activity of liposomal Fenretinide targeted to human neuroblastoma

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. In advanced disease stages, prognosis is poor and treatments have limited efficacy, thus novel strategies are warranted. The synthetic retinoid Fenretinide (HPR) induces apoptosis in NB and melanoma cell lines. We reported an in vitro potentiation of HPR effects on melanoma cells when the drug is incorporated into GD2-targeted immunoliposomes (anti-GD2-SIL-HPR). We investigated the antitumor activity of anti-GD2-SIL-HPR against NB cells, both in vitro and in vivo. Anti-GD2-SIL showed specific, competitive binding to and uptake by, various NB cell lines. In in vitro cytotoxicity studies, NB cells, incubated with 30 microM HPR entrapped in anti-GD2-immunoliposomes, showed a significant reduction in cellular growth compared to free HPR, HPR entrapped in Ab-free liposomes or anti-GD2 empty liposomes. In an in vivo NB metastatic model, we demonstrated that anti-GD2-SIL-HPR completely inhibited the development of macroscopic and microscopic metastases in comparison to controls. Similar, but significantly less potent, antitumor effect was observed also in mice treated with anti-GD2 immunoliposomes without HPR (anti-GD2-SIL-blank) or anti-GD2 MAb alone (p = 0.0297 and p = 0.0294, respectively, vs. anti-GD2-SIL-HPR). Moreover, our results clearly demonstrated that although anti-GD2 MAb had a strong antitumor effect in this in vivo NB model, 100% curability was obtained only after treatment with anti-GD2-SIL-HPR (p < 0.0001). Anti-GD2 liposomal HPR should receive clinical evaluation as adjuvant therapy of neuroblastoma.     

紫花牡荆素体外抑制人宫颈癌HeLa细胞增殖的研究

背景与目的:紫花牡荆素(Casticin)是一种从蔓荆子中提取的具有广泛药理活性的多甲基黄酮化合物.有研究表明Casticin能诱导多种肿瘤细胞凋亡,但其对人宫颈癌细胞的作用尚缺乏文献资料.本研究旨在探讨Casticin抑制体外培养人宫颈癌HeLa细胞增殖作用及其机制.方法:MTT法检测Casticin对人宫颈癌HeLa细胞活性的抑制作用;平皿集落形成法检测Casticin对HeLa细胞集落形成率的影响;流式细胞术(FCM)检测Casticin对HeLa细胞周期的影响;采用Western blot分析蛋白表达的变化.结果:Casticin对人宫颈癌HeLa细胞增殖活性有较强的抑制作用,呈剂量和时间依赖性,作用48 h的IC50值为2.82 μ g/mL;与对照组比较,细胞集落形成率明显下降(P＜0.05);Casticin作用48 h,以浓度依赖方式阻滞HeLa细胞于G2/M期,同时降低Cyclin B1蛋白表达,增高P21蛋白表达.结论:Casticin抑制HeLa细胞的增殖作用可能与其降低Cyclin B1蛋白表达、活化P21蛋白有关.     

Vascular permeability in a human tumour xenograft: molecular charge dependence

Molecular charge is one of the main determinants of transvascular transport. There are, however, no data available on the effect of molecular charge on microvascular permeability of macromolecules in solid tumours. To this end, we measured tumour microvascular permeability to different proteins having similar size but different charge. Measurements were performed in the human colon adenocarcinoma LS174T transplanted in transparent dorsal skinfold chambers in severe combined immunodeficient (SCID) mice. Bovine serum albumin (BSA) and IgG were fluorescently labelled and were either cationized by conjugation with hexamethylenediamine or anionized by succinylation. The molecules were injected i.v. and the fluorescence in tumour tissue was quantified by intravital fluorescence microscopy. The fluorescence intensity and pharmacokinetic data were used to calculate the microvascular permeability. We found that tumour vascular permeability of cationized BSA (pI-range: 8.6-9.1) and IgG (pI: 8.6-9.3) was more than two-fold higher (4.25 and 4.65x10(-7) cm s(-1)) than that of the anionized BSA (pI approximately 2.0) and IgG (pI: 3.0-3.9; 1.11 and 1.93x10(-7) cm s(-1), respectively). Our results indicate that positively charged molecules extravasate faster in solid tumours compared to the similar-sized compounds with neutral or negative charges. However, the plasma clearance of cationic molecules was approximately 2x faster than that of anionic ones, indicating that the modification of proteins enhances drug delivery to normal organs as well. Therefore, caution should be exercised when such a strategy is used to improve drug and gene delivery to solid tumours.     

Mucin impedes cytotoxic effect of 5-FU against growth of human pancreatic cancer cells: overcoming cellular barriers for therapeutic gain

Mucins are high molecular weight glycoproteins expressed on the apical surface of normal epithelial cells. In cancer disease mucins are overexpressed on the entire cellular surface. Overexpression of MUC1 mucin in pancreatic tumours has been correlated with poor patient survival. Current chemotherapeutic approaches such as 5-fluorouracil (5-FU) has produced limited clinical success. In this study we investigated the role of mucin in cytotoxic drug treatment to determine whether the extracellular domain of mucin impedes cytotoxic drug action of 5-FU. Human pancreatic cancer cells revealed high and relatively moderate MUC1 levels for Capan-1 and HPAF-II, respectively, compared to MUC1 negative control (U-87 MG glioblastoma) that showed relatively non-specific anti-MUC1 uptake. Benzyl-alpha-GalNAc (O-glycosylation inhibitor) was used to reduce mucin on cell surfaces, and neuraminidase was used to hydrolyse sialic acid at the distal end of carbohydrate chains. Benzyl-alpha-GalNAc had no effect on cell morphology or proliferation at the concentrations employed. The inhibition of O-glycosylation resulted in significant 5-FU antiproliferative activity against Capan-1 and HPAF-II, but not against U-87 MG. However, the exposure of cells to neuraminidase failed to improve the cytotoxic action of 5-FU. Our experimental findings suggest that the overexpression of mucin produced by human pancreatic tumours might limit the effectiveness of chemotherapy.     

Chemosensitivity profile assay of circulating cancer cells: prognostic and predictive value in epithelial tumors

The prognostic value associated with the detection of circulating tumor cells (CTCs) in metastatic breast cancer by the CellSearch™ technology raise additional issues regarding the biological value of this information. We postulated that a drug‐resistance profile of CTCs may predict response to chemotherapy in cancer patients and therefore could be used for patient selection. One hundred 5 patients with diagnosis of carcinoma were enrolled in a prospective trial. CTCs were isolated from peripheral blood, and positive samples were evaluated for the expression of a panel of genes involved in anticancer drugs resistance. The drug‐resistance profile was correlated with disease‐free survival (DFS; patients in adjuvant setting) and time to progression (TTP; metastatic patients) in a 24‐months follow‐up. Objective response correlation was a secondary end point. Fifty‐one percent of patients were found positive for CTCs while all blood samples from healthy donors were negative. The drug‐resistance profile correlates with DFS and TTP (p < 0.001 in both). Sensitivity of the test: able to predict treatment response in 98% of patients. Specificity of the test: 100%; no sample from healthy subject was positive for the presence of CTCs. Positive and negative predictive values were found to be 96.5 and 100%, respectively. We identified a drug‐resistance profile of CTCs, which is predictive of response to chemotherapy, independent of tumor type and stage of disease. This approach may represent a first step toward the individualization of chemotherapy in cancer patients.     

Interaction between gemcitabine and topotecan in human non-small-cell lung cancer cells: effects on cell survival, cell cycle and pharmacogenetic profile

The pyrimidine analogue gemcitabine is an established effective agent in the treatment of non-small-cell lung cancer (NSCLC). The present study investigates whether gemcitabine would be synergistic with the topoisomerase I inhibitor topotecan against the NSCLC A549 and Calu-6 cells. Cells were treated with gemcitabine and topotecan for 1 h and the type of drug interaction was assessed using the combination index (CI). Cell cycle alterations were analysed by flow cytometry, while apoptosis was examined by the occurrence of DNA internucleosomal fragmentation, nuclear condensation and caspase-3 activation. Moreover, the possible involvement of the PI3K-Akt signalling pathway was investigated by the measurement of Akt phosphorylation. Finally, quantitative, real-time PCR (QRT-PCR) was used to study modulation of the gemcitabine-activating enzyme deoxycytidine kinase (dCK) and the cellular target enzyme ribonucleotide reductase (RR). In results, it was found that simultaneous and sequential topotecan --> gemcitabine treatments were synergistic, while the reverse sequence was antagonistic in both cell lines. DNA fragmentation, nuclear condensation and enhanced caspase-3 activity demonstrated that the drug combination markedly increased apoptosis in comparison with either single agent, while cell cycle analysis showed that topotecan increased cells in S phase. Furthermore, topotecan treatment significantly decreased the amount of the activated form of Akt, and enhanced the expression of dCK (+155.0 and +115.3% in A549 and Calu-6 cells, respectively), potentially facilitating gemcitabine activity. In conclusion, these results indicate that the combination of gemcitabine and topotecan displays schedule-dependent activity in vitro against NSCLC cells. The gemcitabine --> topotecan sequence is antagonistic while drug synergism is obtained with the simultaneous and the sequential topotecan --> gemcitabine combinations, which are associated with induction of decreased Akt phosphorylation and increased dCK expression.     

Fluconazole vs. amphotericin B for the management of candidaemia in adults: a meta-analysis

The incidence of bloodstream infections caused by Candida species is rising. Few published studies have compared the efficacy of fluconazole with that of amphotericin B. We performed a meta-analysis of the prospective studies that compared fluconazole and amphotericin B for the treatment of candidaemia in adults. Data on total mortality, candidaemia-attributable mortality, efficacy, microbiological failure, and toxicity were extracted from eligible studies. All studies appeared homogeneous with respect to the outcome measures. Most patients were at relatively low risk for death as evidenced by the low average physiologic score and the lack of intense immunosuppression. The odds ratios (OR) of treatment with amphotericin B versus fluconazole and 95% confidence intervals (CI) were as follows: total mortality (OR, 1.06; CI, 0.89-1.25), candidaemia-attributable mortality (OR, 1.0; CI, 0.70-1.45), clinical response (OR, 1.14; CI, 0.93-1.39) and microbiological failure according to all Candida species (OR, 0.99; CI, 0.78-1.26). A trend favouring amphotericin B was seen in mycological eradication of non-albicans Candida species (OR, 0.70; CI, 0.47-1.06). Finally, amphotericin B was more toxic than fluconazole (OR, 2.94; CI, 2.14-4.4). In conclusion, fluconazole is as efficacious and less toxic than amphotericin B in stable, not severely immunosuppressed candidaemic patients at low risk for death. However, fluconazole may be less effective than amphotericin B in candidaemias caused by some non-albicans Candida species.     

Comparative studies of chemotherapy of human tumor cells in vitro by tritiated thymidine uptake inhibition and soft agar clonogenic assay

A rapid nonsterile short-term assay has been investigated in which percent inhibition of incorporation of the DNA precursor (3H)-thymidine is measured following exposure of tumor cells to the test drugs. To evaluate the usefulness of the short-term assay in providing a rapid reliable assessment of chemotherapeutic response, the short-term assay was compared with the soft agar clonogenic assay. Sensitivity to five anticancer drugs was compared using three human tumor cell lines (epidermoid carcinoma of the oral cavity, pancreatic carcinoma, and bladder carcinoma). The short-term assay produced results that were similar to results of the clonogenic assay in two of the three tumors tested, for three drugs (cis-platin, doxorubicin, and BCNU), but did not detect responses to two antimetabolites (5-FU and MTX) in any tumor. Further studies of this short-term assay should focus on alkylating agents and other nonantimetabolites.     

碳纳米管携载多柔比星对人卵巢癌耐药细胞体外抑制研究

目的 长期应用多柔比星(doxorubicin,ADM)能够增加药物耐受性及毒副作用,单壁碳纳米管(single-walled carbon nanotubes,SWCNTs)作为药物载体可提高载药率.本研究通过制备携载ADM的碳纳米管复合物然后作用于人卵巢癌ADM耐药细胞株,探讨研究碳纳米管复合物对细胞的杀伤作用.方法 制备ADM/PEG/SWCNTs复合物,傅里叶变换红外光谱扫描仪表征检测PEG/SWCNTs表面官能团的变化;紫外分光光度计测定载药量;WST-1法检测ADM/PEG/SWCNTs对SKOV3/ADM的增殖抑制作用;流式细胞术和Hoechst33258荧光染色观察ADM/PEG/SWC-NTs对细胞凋亡的影响;PCR及蛋白质印迹法检测ADM/PEG/SWCNTs对MDR-1 mRNA及P-gp糖蛋白的表达影响.结果 成功制备ADM/PEG/SWCNTs复合药物,其中ADM在PEG/SWCNTs的载药率为(79.5±10.6)％,包封率(32.0±4.2)％. WST-1实验显示,ADM/PEG/SWCNTs及ADM作用细胞平均IC50为(18.0±1.2)和(37.5±1.9)μg/mL.ADM/PEG/SWCNTs对SKOV3/ADM细胞24、48和72 h细胞增殖抑制率分别为(43.27±3.60)％、(53.40±8.12)％和(68.00±4.26)％,组间比较,差异有统计学意义,P＜0.001.流式细胞术检测ADM/PEG/SWCNTs组及ADM组SKOV3/ADM细胞凋亡率为(14.83±1.86)％和(5.28±0.45)％,F=137.292,P=0.001.Hoechst 33258染色ADM/PEG/SWCNTs组细胞呈明显凋亡形态改变.ADM/PEG/SWCNTs组可下调MDR-1mRNA及P-gp蛋白的表达,经灰度扫描分析,组间比较,差异有统计学意义,F=18.634,P=0.003.结论 体外制备的ADM/PEG/SWCNTs纳米复合物具有高载药率,可抑制人卵巢癌细胞多柔比星耐药细胞增殖,促进人卵巢癌ADM耐药细胞凋亡,可下调MDR 1mRNA及P gp蛋白的表达.     

A novel class of specific Hsp90 small molecule inhibitors demonstrate in vitro and in vivo anti-tumor activity in human melanoma cells

Hsp90 is required for conformational maturation and stability of numerous key signaling proteins (clients) involved in cell proliferation and transformation. Here we describe two novel Hsp90 inhibitors, PF-4470296 and PF-3823863, demonstrate a differential sensitivity in B-Raf mutant (V600E) versus wild-type protein degradation. These two inhibitors inhibit proliferation and anchorage-independent growth, and abolish in vivo xenograft tumor growth in melanoma cells regardless of B-Raf mutation status. Mutant B-Raf protein and other Hsp90 clients, such as cMet, ErbB2, C-Raf, and AKT, are degraded in cells and xenograft tumors. Our results indicate that Hsp90 inhibitors induce anti-tumor activity in melanoma cells and are likely to show therapeutic benefit in melanoma patients by collaboratively targeting multiple pathways.     

通关藤提取物体外对人正常免疫细胞及造血干细胞的影响

目的　观察通关藤提取物（消癌平注射液）对正常免疫细胞及造血干细胞功能的影响。方法　采用ＭＴＴ法检测通关藤提取物对正常人淋巴细胞以及ＣｏｎＡ、ＬＰＳ诱导的人淋巴细胞增殖活性的影响,中性红法检测巨噬细胞的吞噬功能,ＣＦＵ-ＧＭ半固体集落培养法检测造血干细胞的集落形成能力。结果　通关藤提取物体外对ＣｏｎＡ诱导的正常淋巴细胞和ＬＰＳ诱导的正常淋巴细胞有促增殖作用且呈剂量依赖性。通关藤提取物对正常巨噬细胞吞噬中性红染液的能力无明显影响。对正常骨髓／外周血造血干细胞ＣＦＵ-ＧＭ的形成没有显著影响。结论　通关藤提取物体外对正常免疫细胞和造血干细胞无明显细胞毒作用,并且有促进Ｔ、Ｂ细胞的增殖作用。     

JaponiconeA induces apoptosis of bortezomib-sensitive and -resistant myeloma cells in vitro and in vivo by targeting IKKβ

Objective:Multiple myeloma (MM) remains incurable with high rates of relapse. New therapeutic drugs are therefore urgently needed to improve the prognosis. JaponiconeA (JA), a natural product isolated from Inula japonica Thunb, has shown good anti-MM potential. A comprehensive study should therefore be conducted to identify both the in vitro and in vivo mechanisms of the anti-MM effects of JA.Methods:CCK8 assays and flow cytometry were used to detect the proliferation, apoptosis, and cell cycle of MM cell lines when treated with JA. In vivo experiments were conducted using subcutaneous xenograft mouse models. We also identified possible targets and the mechanism of JA using RNA-seq and c-Map databases, and identified the specific targets of JA in bortezomib-sensitive and -resistant MM cell lines using CETSA, DARTS, and rescue experiments. Furthermore, JA and bortezomib were used separately or together to characterize their possible synergistic effects.Results:In vitro, JA inhibited proliferation, and induced apoptosis and G2/M phase arrest in MM cell lines, and selectively killed primary CD138⁺ MM cells. In vivo, JA also demonstrated a strong anti-tumor effect with no observable toxicity. In addition, JA showed synergetic effects in combination with bortezomib, and enhanced the anti-tumor effect of bortezomib in bortezomib-resistant cells. CETSA and DARTS confirmed direct binding of JA to NF-κB inhibitor kinase beta (IKKβ), and overexpression of IKKβ or knockdown of IκBα partially rescued the apoptosis induced by JA.Conclusions:JA exhibited strong anti-tumor effects in MM. It sensitized myeloma cells to bortezomib and overcame NF-κB-induced drug resistance by inhibiting IKKβ, providing a new treatment strategy for MM patients.