The association of carbohydrate antigen 19-9 response with radiologic response and survival in intrahepatic cholangiocarcinoma: A prospective cohort study.

BACKGROUND: The role of carbohydrate antigen 19-9 (CA 19-9) in response assessment among patients with intrahepatic cholangiocarcinoma (iCCA) remains unknown. The authors studied the association of the CA 19-9 response (defined as a reduction >50% from baseline) with the radiologic response and the outcome in patients with unresectable iCCA. METHODS: A prospective cohort of 422 patients who were initially diagnosed with unresectable iCCA, had baseline CA 19-9 levels ≥100 U/mL, and received treatment with systemic therapies at the authors' institution between January 2017 and December 2021 were enrolled in this study. The radiologic response was assessed using the Response Evaluation Criteria in Solid Tumors version 1.1. A landmark assessment of the CA 19-9 response and the radiologic response was performed. The associations between CA 19-9 response and imaging response, progression-free survival (PFS), and overall survival (OS) were analyzed. RESULTS: Two hundred sixty-seven patients (63.3%) had a CA 19-9 response. A CA 19-9 response was observed in 123 of 132 (93.2%) radiologic responders and in 144 of 290 (49.7%) radiologic nonresponders (p < .001). CA 19-9 responders outperformed nonresponders in median PFS (10.6 vs. 3.6 months; hazard ratio [HR], 4.8 months; 95% confidence interval [CI], 3.8-6.0 months; p < .001) and OS (21.4 vs. 6.3 months; HR, 5.3 months; 95% CI, 4.2-6.7 months; p < .001). The common independent predictors of both OS and PFS included metastasis, CA 19-9 nonresponder status, and radiologic nonresponder status in multivariable analysis. CONCLUSIONS: CA 19-9 response is a valuable addition to assess tumor response and is associated with improved outcomes in patients with iCCA. Achieving a CA 19-9 response should be one of the therapeutic objectives of patients with iCCA after systemic therapies. PLAIN LANGUAGE SUMMARY: A decline in carbohydrate antigen 19-9 levels from elevated baseline levels should be one of the therapeutic aims of patients with intrahepatic cholangiocarcinoma who are managed with systemic therapies.

Clinical and biomarker analyses of sintilimab plus gemcitabine and cisplatin as first-line treatment for patients with advanced biliary tract cancer.

The prognosis of biliary tract cancer (BTC) remains unsatisfactory. This single-arm, phase II clinical trial (ChiCTR2000036652) investigated the efficacy, safety, and predictive biomarkers of sintilimab plus gemcitabine and cisplatin as the first-line treatment for patients with advanced BTCs. The primary endpoint was overall survival (OS). Secondary endpoints included toxicities, progression-free survival (PFS), and objective response rate (ORR); multi-omics biomarkers were assessed as exploratory objective. Thirty patients were enrolled and received treatment, the median OS and PFS were 15.9 months and 5.1 months, the ORR was 36.7%. The most common grade 3 or 4 treatment-related adverse events were thrombocytopenia (33.3%), with no reported deaths nor unexpected safety events. Predefined biomarker analysis indicated that patients with homologous recombination repair pathway gene alterations or loss-of-function mutations in chromatin remodeling genes presented better tumor response and survival outcomes. Furthermore, transcriptome analysis revealed a markedly longer PFS and tumor response were associated with higher expression of a 3-gene effector T cell signature or an 18-gene inflamed T cell signature. Sintilimab plus gemcitabine and cisplatin meets pre-specified endpoints and displays acceptable safety profile, multiomics potential predictive biomarkers are identified and warrant further verification.

Use of miRNA Sequencing to Reveal Hub miRNAs and the Effect of miR-582-3p/SMAD2 in the Progression of Hepatocellular Carcinoma.

Hepatocellular carcinoma is a common tumor with a high fatality rate worldwide, and exploring its pathogenesis and deterioration mechanism is a focus for many researchers. Increasing evidence has shown that miRNAs are involved in the occurrence and progression of a variety of cancers, including hepatocellular carcinoma. Therefore, this study mainly aimed identify key miRNAs related to hepatocellular carcinoma and explore their potential functions and clinical significance. In this study, we performed miRNA sequencing on three pairs of hepatocellular carcinoma tissue samples and screened 26 differentially expressed miRNAs. Then 2 key miRNAs (miR-139-5p and miR-582-3p) were screened by Kaplan-Meier curve analysis, Cox multivariate analysis and qPCR methods. The expression of miR-582-3p was positively correlated with clinicopathological parameters in patients with hepatocellular carcinoma. Subsequently, miRwalk and starbase were used to predict the target genes of key miRNAs, and then the key pairs miR-582-3p/SMAD2 identified by WGCNA, PPI, qPCR and Pearson correlation analysis. Finally, a dual luciferase experiment, the rescue-of-function experiment and qPCR confirmed that miR-582-3p directly targets SMAD2 and regulates the proliferation, migration and invasion of HepG2 cells by targeting SMAD2. At the same time, interference with SMAD2 can influence the effect of miR-582-3p on HepG2 cells. In conclusion, our findings confirm that miR-582-3p is an independent factor for the prognosis of hepatocellular carcinoma patients, and can regulate the progression of hepatocellular carcinoma cells by targeting SMAD2.

Hydrostatic pressure induces osteogenic differentiation of adipose-derived mesenchymal stem cells through increasing lncRNA-PAGBC.

Induced osteogenesis of adipose-derived mesenchymal stem cells (AMSCs) has been used to facilitate bone regeneration. Specifically, hydrostatic pressure (HP) has been implicated as a key regulator of AMSC differentiation, whereas the mechanisms that underlie the effects of HP on osteogenesis of AMSCs are not fully understood. Long noncoding RNAs (lncRNAs) are emerging regulators for osteogenic differentiation from AMSCs. In the current study, we found that lncRNA-PAGBC was a specific lncRNA that significantly upregulated during osteogenic differentiation of AMSCs based on published database. HP increased lncRNA-PAGBC, which is a competitive endogenous RNA (ceRNA) that binds to the osteogenesis-inhibitory microRNA, miR-133b, to regulate osteogenic differentiation of AMSCs. Moreover, a key osteogenesis-trigger gene, runt-related transcription factor 2 (RUNX2), was identified as a target gene for miR-133b. Suppression of RUNX2 by miR-133b caused impaired osteogenic differentiation of AMSCs. Furthermore, lncRNA-PAGBC overexpression upregulated, whereas lncRNA-PAGBC silencing decreased the expression of RUNX2 through miR-133b. Together, these data suggest that HP induces osteogenic differentiation of AMSCs through increasing lncRNA-PAGBC.

Macro-Microporous Surface with Sulfonic Acid Groups and Micro-Nano Structures of PEEK/Nano Magnesium Silicate Composite Exhibiting Antibacterial Activity and Inducing Cell Responses.

PURPOSE: To improve the surface bio-properties of polyetheretherketone (PEEK)/nano magnesium silicate (n-MS) composite (PC). MATERIALS AND METHODS: The surface of PC was firstly treated by particle impact (PCP) and subsequently modified by concentrated sulfuric acid (PCPS). RESULTS: PCPS surface exhibited not only macropores with sizes of about 150 µm (fabricated by particle impact) but also micropores with sizes of about 2 µm (created by sulfonation of PEEK) on the macroporous walls, and sulfonic acid (-SO3H) groups were introduced on PCPS surface. In addition, many n-MS nanoparticles were exposed on the microporous walls, which formed micro-nano structures. Moreover, the surface roughness and hydrophilicity of PCPS were obviously enhanced as compared with PC and PCP. Moreover, the apatite mineralization of PCPS in simulated body fluid (SBF) was obviously improved as compared with PC. Furthermore, compared with PC and PCP, PCPS exhibited antibacterial performances due to the presence of -SO3H groups. In addition, the responses (eg, adhesion and proliferation as well as differentiation) of bone marrow mesenchymal stem cell of rat to PCPS were significantly promoted as compared with PC and PCP. CONCLUSION: PCPS with macro-microporous surface containing -SO3H groups and micro-nano structures exhibited antibacterial activity and induced cell responses, which might possess large potential for bone substitute and repair.

Genome-wide characterization of lncRNAs in acute myeloid leukemia.

Long noncoding RNAs (lncRNAs) are a large family of noncoding RNAs that play a critical role in various normal bioprocesses as well as tumorigenesis. However, the expression patterns and biological functions of lncRNAs in acute leukemia have not been well studied. Here, we performed transcriptome-wide lncRNA expression profiling of acute myeloid leukemia (AML) patient samples, along with non-leukemia control hematopoietic samples. We found that lncRNAs were differentially expressed in AML samples relative to control samples. Notably, we identified that lncRNAs upregulated in AML (relative to the control samples) are associated with a lower degree of DNA methylation and a higher ratio of being bound by transcription factors such as SP1, STAT4, ATF-2 and ELK-1 compared with those downregulated in AML. Moreover, an enrichment of H3K4me3 and a depletion of H3K27me3 were observed in upregulated lncRNAs in AML. Expression patterns of three types of lncRNAs (antisense, enhancer and intergenic lncRNAs) have previously been characterized. Of the identified lncRNAs, we found that high expression level lncRNA LOC285758 is associated with the poor prognosis in AML patients. Furthermore, we found that LOC285758 regulates proliferation of AML cell lines by enhancing the expression of HDAC2, a key factor in carcinogenesis. Collectively, our study depicts a landscape of important lncRNAs in AML and provides novel potential therapeutic targets and prognostic markers for AML treatment.

Zein regulating apatite mineralization, degradability, in vitro cells responses and in vivo osteogenesis of 3D-printed scaffold of n-MS/ZN/PCL ternary composite.

Bioactive and degradable scaffolds of nano magnesium silicate (n-MS)/zein (ZN)/poly(caprolactone) (PCL) ternary composites were prepared by 3D-printing method. The results showed that the 3D-printed scaffolds possessed controllable pore structure, and pore morphology, pore size, porosity and pore interconnectivity of the scaffolds can be efficiently adjusted. In addition, the apatite-mineralization ability of the scaffolds in simulated body fluids was obviously improved with the increase of ZN content, in which the scaffold with 20 w% ZN (C20) possessed excellent apatite-mineralization ability. Moreover, the degradability of the scaffolds was significantly enhanced with the increase of ZN content in the scaffolds. The degradation of ZN produced acidic products that could neutralize the alkaline products from the degradation of n-MS, which avoid the increase of pH value in degradable solution. Furthermore, the MC3T3-E1 cells responses (e.g. proliferation and differentiation, etc.) to the scaffolds were significantly promoted with the increase of ZN content. The in vivo osteogenesis of the scaffolds implanted the femur defects of rabbits was investigated by micro-CT and histological analysis. The results demonstrated that the new bone formation was significantly enhanced with the increase of ZN content, in which the C20 scaffold induced the highest new bone tissues, indicating excellent osteogenesis. The results suggested that the ZN in the ternary composite scaffolds played key roles in assisting bone regeneration in vivo.

[Effect of CP Metronomic Chemotherapy on RPMI 8226 Cell Proli-feration and Notch1/NF-κB Signaling Pathway In Vitro].

OBJECTIVE: To investigate the effect of metronomic chemotherapy of low dose phosphoramide combined with prednisolone (CP metronomic chemotherapy) on proliferation and apoptosis of RPMI 8226 cells, and to explore its regulating effect on Notch1/NF-κB signaling pathways. METHODS: Experiment was divided into the DMSO control group, and the phosphoramide mustard (PM) group, the prednisolone group, the phosphoramide mustard plus prednisolone group (the CP group). RPMI 8226 cells were treated with different drugs, CCK-8 method was used to detect cell proliferation, flow cytometry was used to detect the cell cycle and apoptosis, reverse transcription PCR was used to detect Notch1 and NF-κB mRNA expression level. RESULTS: Compared with DMSO control group, RPMI8226 cell proliferation inhibition rate in all the PM, prednisolone and CP groups increased significantly with prolonging of time (r of 0.994,0.996,0.999, respectively, P<0.001). And at the same time, the inhibitory rate of cell proliferation was significantly different; the cell inhibitory rate in PM group was lowest, that in CP group was highgest, that in prednissone group was intermediate (P<0.01). After 48 hours, compared with the DMSO control group, the G1/G0 cell proportion in treatment group increased significantly, S phase cell proportion decreased significantly, especially in PM and CP groups. The G2/M phase cell proportion increased in PM group, while reduced in the prednisolone and the CP groups. After 48 hours, compared with the DMSO control group, RPMI 8226 cell apoptosis rate increased as follow: in PM, pre-dnisolone and CP group(P<0.01). After 48 hours, compared with the DMSO control group, Notch1 and NF-κB mRNA expression in the prednisolone, the PM and the CP group decreased significantly(P<0.001). CONCLUSION: CP metronomic chemotherapy can significantly reduce RPMI 8226 cell proliferation, promote RPMI 8226 cell apoptosis, arrest RPMI 8226 cells mainly in the G1/G0 phase, and significantly reduce Notch1 and NF-κB expression levels. It is suggested that Notch1/NF-κB signaling pathways is involved in CP metronomic chemotherapy for MM.

A mercuric ensemble based on a cycloruthenated complex as a visual probe for iodide in aqueous solution.

A new water-soluble cycloruthenated complex Ru(bthiq)(dcbpy)2(+) (1, Hbthiq=1-(2-benzo[b]thiophenyl)isoquinoline, dcbpy=4,4'-dicarboxylate-2,2'-bipyridine) was designed and synthesized to form its mercuric ensemble (1-Hg(2+)) to achieve visual detection of iodide anions. The binding constant of 1-Hg(2+) is calculated to be 2.40×10(4)M(-1), which is lower than that of HgI2. Therefore, the addition of I(-) to the aqueous solution of 1-Hg(2+)lead to significant color changes from yellow to deep-red by the release of 1. The results showed that iodide anions could be easily detected by the naked eyes. The detection limit of iodide anion is calculated as 0.77µM. In addition, an easily-prepared test strip of 1-Hg(2+) was obtained successfully to detect iodide anions.

Degradability, bioactivity, and osteogenesis of biocomposite scaffolds of lithium-containing mesoporous bioglass and mPEG-PLGA-b-PLL copolymer.

Biocomposite scaffolds of lithium (Li)-containing mesoporous bioglass and monomethoxy poly(ethylene glycol)-poly(D,L-lactide-co-glycolide)-poly(L-lysine) (mPEG-PLGA-b-PLL) copolymer were fabricated in this study. The results showed that the water absorption and degradability of Li-containing mesoporous bioglass/mPEG-PLGA-b-PLL composite (l-MBPC) scaffolds were obviously higher than Li-containing bioglass/mPEG-PLGA-b-PLL composite (l-BPC) scaffolds. Moreover, the apatite-formation ability of l-MBPC scaffolds was markedly enhanced as compared with l-BPC scaffolds, indicating that l-MBPC scaffolds containing mesoporous bioglass exhibited good bioactivity. The cell experimental results showed that cell attachment, proliferation, and alkaline phosphatase activity of MC3T3-E1 cells on l-MBPC scaffolds were remarkably improved as compared to l-BPC scaffolds. In animal experiments, the histological elevation results revealed that l-MBPC scaffolds significantly promoted new bone formation, indicating good osteogenesis. l-MBPC scaffolds with improved properties would be an excellent candidate for bone tissue repair.

[Efficacy of continuous low-dose cyclophosphamide and prednisone regimen in refractory multiple myeloma patients with severe heart failure].

OBJECTIVE: To observe treatment response, survival, safety and the improvement of ECOG in patients with refractory multiple myeloma (MM) with serious heart failure after the administration of continuous low-dose of cyclophosphamide combined with prednisone (CP). METHODS: From January 2005 to September 2013, a total of 75 patients were treated by metronomic chemotherapy with continuous low-dose cyclophosphamide (50 mg/d) and prednisone (15 mg/d). RESULTS: Among the 75 patients, 2 were lost for follow-up. In the 73 available patients, the overall response was 64.4%, including 2 patients (2.7%) with complete remission (CR), 4 cases (5.5%) very good partial remission (VGPR), and 24 patients (32.9%) partial remission (PR). The median survival was 12 months (1-70 months) with a median onset time of 90 days (16-120) and a median progressive freedom survival of 12 months (1-60). The level of B-type natriuretic peptide in responders declined significantly, as compared to no responders [(336.6 ± 30.3) ng/L vs (906.4 ± 104.8) ng/L, P<0.01]. Common adverse events were as follows: 32 (43.8%) cases of bone marrow suppression, 26 (35.6%) cases of infection, 8 cases of dizzy as well as sleepiness (11.0%), 7(9.6%) cases of Cushing syndrome, 4 (5.5%) cases of secondary diabetes mellitus, and 3 (4.1%) cases of edema respectively. CONCLUSION: The metronomic chemotherapy of cyclophosphamide combined with prednisone had satisfactory impact on the treatment outcome, including the improvement of cardiac functions and physical capacities, better survival and safety in refractory MM with serious heart failure. It provides a novel regimen for such patients.

Bioactive and degradable scaffolds of the mesoporous bioglass and poly(l-lactide) composite for bone tissue regeneration.

Bioactive scaffolds of the mesoporous bioglass (m-BG) and poly(l-lactide) (PLLA) composite were fabricated using a solvent casting-particulate leaching method. The results showed that incorporation of the m-BG into PLLA significantly improved the in vitro water absorption, degradability and apatite-formation ability of the m-BG-PLLA composite scaffolds, which were m-BG content dependent. Moreover, addition of the m-BG into PLLA could neutralize the acidic degradation products of PLLA and thus compensate for the decrease of the pH value. In cell culture experiments, the results revealed that the m-BG-PLLA composite scaffolds enhanced attachment, proliferation and alkaline phosphatase (ALP) activity of MC3T3-E1 cells, which were m-BG content dependent. In animal experiments, the SRmCT and histological elevation results showed that the composite scaffolds significantly improved osteogenesis in vivo. It can be suggested that incorporation of bioactive materials of m-BG into PLLA was a useful approach to obtain composite scaffolds with improved properties (such as water absorption, degradability, bioactivity and osteogenesis), and the composite scaffolds with excellent biocompatibility could be promising bioactive implants for bone regeneration.

Mesoporous magnesium silicate-incorporated poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) bioactive composite beneficial to osteoblast behaviors.

Mesoporous magnesium silicate (m-MS) and poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCL-PEG-PCL) composite (m-MPC) was synthesized by solvent casting method. The results suggest that the mechanical properties of compressive strength and elastic modulus, as well as hydrophilicity, of the m-MPC increased with increase of m-MS content in the composites. In addition, the weight loss of the m-MPC improved significantly with the increase of m-MS content during composite soaking in phosphate-buffered saline for 10 weeks, indicating that incorporation of m-MS into PCL-PEG-PCL could enhance the degradability of the m-MPC. Moreover, the m-MPC with 40 w% m-MS could induce a dense and continuous apatite layer on its surface after soaking in simulated body fluid for 5 days, which was better than m-MPC 20 w% m-MS, exhibiting excellent in vitro bioactivity. In cell cultural experiments, the results showed that the attachment and viability ratio of MG63 cells on m-MPC increased significantly with the increase of m-MS content, showing that the addition of m-MS into PCL-PEG-PCL could promote cell attachment and proliferation. The results suggest that the incorporation of m-MS into PCL-PEG-PCL could produce bioactive composites with improved hydrophilicity, degradability, bioactivity, and cytocompatibility.

Continuous low-dose cyclophosphamide and prednisone in the treatment of relapsed/refractory multiple myeloma with severe heart failure.

Patients with relapsed/refractory (R/R) multiple myeloma (MM) complicated by severe heart failure typically do not tolerate conventional chemotherapy. Our previous study indicated that R/R MM patients with severe comorbidities could benefit from continuous low-dose oral cyclophosphamide and prednisone (CP regimen). We hereby performed a study of 56 R/R MM patients with severe heart failure (New York Heart Association class ≥ III) receiving the treatment of CP regimen. Among the 54 evaluable patients, clinical benefit was noted in 63.0% (complete response, 3.7%; very good partial response, 7.4%; partial response, 48.1%; stable disease, 3.7%). The median overall survival (OS) and progression-free survival (PFS) were 8 and 6 months. The left ventricular ejection fraction (LVEF) and the serum levels of B-type natriuretic peptide (BNP) were improved significantly. Slight adverse events were observed. In summary, the CP regimen is effective in R/R MM patients complicated with severe heart failure.

[Anti-angiogenesis effect of metronomic chemotherapy in multiple myeloma patients].

OBJECTIVE: To investigate effects of low-dose cyclophosphamide and prednisone (CP) metronomic chemotherapy on microvessel density of bone marrow, serum vascular endothelial growth factor (VEGF) and platelet derived growth factor BB (PDGF-BB)in multiple myeloma (MM) patients. METHODS: 54 refractory or relapsed MM patients were treated with CP metronomic chemotherapy consisted of oral cyclophosphamide (CTX, 50 mg/d) and prednisone (Pred, 15 mg/d). Bone marrow and peripheral blood of each patient were collected before and 2, 4, 6 months after treatment. Among the 37 assessable patients, 30 cases were responsive with the response rate of 81.08%. Another 17 cases were follow-uped less than 6 months or failure to obtain serum samples or lost to follow-up. Microvessel density of bone marrow was measured by immunohistochemistry and serum VEGF/PDGF-BB expression was analyzed by ELISA in the 37 assessable patients. RESULTS: 2, 4, 6 months following CP metronomic chemotherapy, microvessel densities of bone marrow in the responders were 33.1 ± 4.8/HP, 24.8 ± 3.7/HP, 19.7 ± 2.1/HP respectively; the expressions of VEGF were (394 ± 57) ng/L, (268 ± 32) ng/L and (217 ± 20) ng/L respectively; the expressions of PDGF-BB were (304 ± 31) ng/L, (274 ± 31) ng/L and (196 ± 22) ng/L respectively. After CP metronomic chemotherapy, there were significantly lower of microvessel density, VEGF and PDGF-BB levels than pretreatment \[MVD 48.5 ± 5.9/HP, VEGF (517 ± 60) ng/L, PDGF-BB (484 ± 60) ng/L\]in the responders (P < 0.01). While in the non-responders, after treated by CP metronomic chemotherapy for 2 months, microvessel density, the expression of VEGF and the expression of PDGF-BB were 32.5 ± 4.7/HP, 512 ± 39 ng/L and (452 ± 39) ng/L respectively. There were no significant changes of MVD, VEGF and PDGF-BB levels compared with pretreatment \[MVD 33.2 ± 5.6/HP,VEGF (498 ± 55) ng/L, PDGF-BB (488 ± 44) ng/L\] (P > 0.05). CONCLUSIONS: Our findings suggested that continuous low-dose CP metronomic chemotherapy could decrease microvessel density of bone marrow in MM patients. Furthermore, it down-regulated expression of serum VEGF and PDGF-BB to exert its anti-angiogenesis in MM.

A combination of thalidomide and arsenic trioxide is effective and well tolerated in patients with myelodysplastic syndromes.

Twenty-two patients with myelodysplastic syndromes (MDS) were treated with thalidomide plus arsenic trioxide (ATO). Twenty-two MDS patients receiving supportive care were used as controls. The remission was achieved in 4 patients (18.2%) receiving thalidomide/ATO, and none in the control group (p<0.05). Fifteen of 22 patients in the treatment group achieved hematologic improvement (68.2% vs. 27.3% in the control, p<0.05). The progression-free survival was longer in the treatment group than that in the control (26 vs. 10 months, p<0.05). The overall survival was also longer in the treatment group than that in the control (36 vs. 16 months, p<0.05). No severe adverse reactions were observed. These preliminary findings suggest that thalidomide/ATO combination treatment is effective and safe for MDS.

Continuous administration of low-dose cyclophosphamide and prednisone as a salvage treatment for multiple myeloma.

PURPOSE: The purpose of this study was to evaluate the efficacy and tolerability of continuous low-dose cyclophosphamide and prednisone (CP) as a salvage therapy for multiple myeloma (MM). PATIENTS AND METHODS: A total of 27 consecutive patients with MM received a treatment regimen that consisted of oral cyclophosphamide 50 mg and prednisone 15 mg daily. Nineteen patients had severe comorbid conditions; 8 were unwilling to continue conventional chemotherapy as a result of severe infection associated with the conventional chemotherapy. Patients had received 1 to 4 chemotherapeutic regimens before the enrollment. RESULTS: The overall response rate (complete remission, very good partial response, and partial response) was 66.7%. The median time to response was 2 months. In the patients who responded to the treatment, the median progression-free survival (PFS) has not been reached. In the nonresponding patients, the median PFS was 4 months. CONCLUSION: Continuous low-dose CP is an effective and well-tolerated salvage therapy for MM.

Overexpression of the PSMB5 gene contributes to bortezomib resistance in T-lymphoblastic lymphoma/leukemia cells derived from Jurkat line.

OBJECTIVE: To study the mechanism of bortezomib resistance in JurkatB lines derived from T-lymphoblastic lymphoma/leukemia Jurkat line. MATERIALS AND METHODS: Cytotoxicities of popular chemotherapeutic drugs to JurkatB cells were analyzed by trypan blue assay. Functional drug efflux in JurkatB cells was determined by flow cytometry utilizing daunorubicin and the expression of P-glycoprotein (P-gp) was detected by Western blot. mRNA expression levels of proteasome beta5 subunit (PSMB5) were measured by quantitation real-time reverse transcription polymerase chain reaction. In situ hybridization was performed to detect the amplification of PSMB5 gene. The chymotrypsin-like activities were assayed by measuring the release of the fluorescent 7-amido-4-methylcoumarin (AMC) from the substrate N-succinyl-Leu-Leu-Val-Tyr-AMC. Cytogenetic studies were performed using R-banded metaphases and fluorescence in situ hybridization (FISH) analysis. IkappaB-alpha levels were detected by Western blot. RESULTS: No cross-resistance to daunorubicin, adriamycin, vindesine, and etoposide was found in JurkatB cells. No evidence of drug efflux was found in JurkatB cells and the expression of P-gp was negative. The PSMB5 mRNA was overexpressed in highly resistant JurkatB5 and JurkatB1 lines compared with parental Jurkat, corresponding well with the increase of chymotrypsin-like activity and a karyotype of i(14q). Amplification of PSMB5 gene was demonstrated by in situ hybridization and FISH. The decreased IkappaB-alpha level in JurkatB5 cells after bortezomib treatment indicating an upregulation of nuclear factor-kappaB (NF-kappaB) activity. CONCLUSION: The mechanism of bortezomib resistance is different from that of multidrug resistance. Overexpression of PSMB5 is an important mechanism for bortezomib resistance in JurkatB lines. NF-kappaB may play a critical role in evading the apoptotic effects.

Point mutation of the proteasome beta5 subunit gene is an important mechanism of bortezomib resistance in bortezomib-selected variants of Jurkat T cell lymphoblastic lymphoma/leukemia line.

To study the mechanism of acquired resistance to bortezomib, a new antitumor drug that is the first therapeutic proteasome inhibitor, we established a series of bortezomib-resistant T lymphoblastic lymphoma/leukemia cell lines, designated the JurkatBs, from the parental Jurkat line via repeated drug selection. There were no significant differences in the growth curves or colony formation between the JurkatB cells and parental Jurkat cells. The effects of bortezomib on cytotoxicity, cell cycle arrest, and induction of apoptosis were decreased in JurkatB cells compared with parental Jurkat cells. A mutation in the proteasome beta5 subunit (PSMB5) gene (G322A), which encodes an amino acid change from Ala to Thr at polypeptide position 108, was detected by sequencing full-length cDNA clones and direct polymerase chain reaction products of the PSMB5 gene. Bortezomib caused less inhibition of chymotrypsin-like activity in resistant cells. When the G322A mutant PSMB5 was retrovirally introduced into parental Jurkat cells, it conferred bortezomib resistance to these cells, resulting in decreased cytotoxicity, apoptosis, and inhibition of chymotrypsin-like activity. The predicted structure of A108T-mutated PSMB5 shows a conformational change that suggests decreased affinity to bortezomib. In short, the G322A mutation of the PSMB5 gene is a novel mechanism for bortezomib resistance.