Construction of a recombinant lentivirus-mediated shRNA expression vector targeting the human PSMD10 gene and validation of RNAi efficiency in RPMI-8226 multiple myeloma cells

This study aimed to investigate the mechanism of betulinic acid on multiple myeloma cell resistance to bortezomib. To this end, the bortezomib-resistant RPMI-8226-R cells were generated by prolonged treatment of RPMI-8226 cells with increasing concentrations of bortezomib. Based on the measurements of cell viability and colony number, RPMI-8226-R cells exhibited enhanced resistance to bortezomib than RPMI-8226 cells. Treatment with betulinic acid resulted in increased sensitivity of RPMI-8226-R to bortezomib. When RPMI-8226-R cells were co-treated with bortezomib and betulinic acid, there was an increase in apoptosis rate, cleaved caspase-3, cleaved caspase-9 expression and the decrease in p-AKT/AKT and p-mTOR/mTOR levels. These results suggest that betulinic acid enhances the sensitivity of RPMI-8226-R cells to bortezomib by inhibiting the activation of the AKT/mTOR pathway in bortezomib-resistant multiple myeloma cells.

Download Full-text

Reduced Response of IRE1α/Xbp-1 Signaling Pathway to Bortezomib Contributes to Drug Resistance in Multiple Myeloma Cells

Tumori Journal ◽

10.5301/tj.5000554 ◽

2016 ◽

Vol 103 (3) ◽

pp. 261-267 ◽

Cited By ~ 4

Author(s):

Xiaoxuan Xu ◽

Junru Liu ◽

Beihui Huang ◽

Meilan Chen ◽

Shiwen Yuan ◽

...

Keyword(s):

Multiple Myeloma ◽

Drug Resistance ◽

Signaling Pathway ◽

Cell Lines ◽

Resistance Mechanism ◽

Proteasome Inhibition ◽

Myeloma Cells ◽

Potential Marker ◽

Rpmi 8226 ◽

Basic Level

Purpose Proteasome inhibition with bortezomib eliminates multiple myeloma (MM) cells by partly disrupting unfolded protein response (UPR). However, the development of drug resistance limits its utility and resistance mechanism remains controversial. We aimed to investigate the role of IRE1α/Xbp-1 mediated branch of the UPR in bortezomib resistance. Methods The expression level of Xbp-1s was measured in 4 MM cell lines and correlated with sensitivity to bortezomib. LP1 and MY5 cells with different Xbp-1s level were treated with bortezomib; then pivotal UPR regulators were compared by immunoblotting. RPMI 8226 cells were transfected with plasmid pEX4-Xbp-1s and exposed to bortezomib; then apoptosis was determined by immunoblotting and flow cytometry. Bortezomib-resistant myeloma cells JJN3.BR were developed and the effect on UPR signaling pathway was determined. Results By analyzing 4 MM cell lines, we found little correlation between Xbp-1s basic level and bortezomib sensitivity. Bortezomib induced endoplasmic reticulum stress-initiated apoptosis via inhibiting IRE1α/Xbp-1 pathway regardless of Xbp-1s basic level. Exogenous Xbp-1s reduced cellular sensitivity to bortezomib, suggesting the change of Xbp-1s expression, not its basic level, is a potential marker of response to bortezomib in MM cells. Furthermore, sustained activation of IRE1α/Xbp-1 signaling pathway in JJN3.BR cells was identified. Conclusions Our data indicate that reduced response of IRE1α/Xbp-1 signaling pathway to bortezomib may contribute to drug resistance in myeloma cells.

Download Full-text

Constructing a Novel Hypoxia-Inducible Bidirectional shRNA Expression Vector for Simultaneous Gene Silencing in Colorectal Cancer Gene Therapy

Cancer Biotherapy & Radiopharmaceuticals ◽

10.1089/cbr.2017.2401 ◽

2018 ◽

Vol 33 (3) ◽

pp. 118-123 ◽

Cited By ~ 1

Author(s):

Bita Javan ◽

Majid Shahbazi

Keyword(s):

Colorectal Cancer ◽

Gene Therapy ◽

Gene Silencing ◽

Expression Vector ◽

Cancer Gene Therapy ◽

Cancer Gene ◽

Shrna Expression ◽

Shrna Expression Vector

Download Full-text

Anti-Tumor Activity of IPI-504, a Novel Hsp90 Inhibitor in Multiple Myeloma.

Blood ◽

10.1182/blood.v104.11.4922.4922 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4922-4922 ◽

Cited By ~ 2

Author(s):

Vito J. Palombella ◽

Emmanuel Normant ◽

Janid Ali ◽

John Barrett ◽

Michael Foley ◽

...

Keyword(s):

Multiple Myeloma ◽

Tumor Cells ◽

Aqueous Solubility ◽

Hsp90 Inhibitor ◽

Tumor Xenograft ◽

Active Metabolites ◽

Myeloma Cells ◽

Rpmi 8226

Abstract IPI-504 is a novel inhibitor of Hsp90 based on the geldanamycin pharmacophore. When placed in rat, monkey, and human blood, IPI-504 rapidly converts to the known and well-studied compound 17-allylamino-17-demethoxy-geldanamycin (17-AAG). 17-AAG is the subject of multiple clinical trials for the treatment of hematologic and solid tumors. However, 17-AAG suffers from poor aqueous solubility necessitating the use of sub-optimal formulations to deliver this agent to patients. IPI-504 is over 1000-fold more soluble than 17-AAG in aqueous solution. In vitro, both 17-AAG and IPI-504 bind tightly to, and selectively inhibit Hsp90 derived from cancer cells. The cytotoxic effect of IPI-504, as well as its ability to stimulate the degradation of Hsp90 client proteins and increase the intracellular levels Hsp70, were monitored in two human multiple myeloma cells lines (RPMI-8226 and MM1.S). The effects of IPI-504 were compared to 17-AAG. We demonstrate that the actions of IPI-504 are bioequivalent to 17-AAG and that both compounds induce apoptosis in these cells and stimulate the degradation of HER2 and c-Raf. In addition, both agents stimulate Hsp70 protein levels. In all cases the EC50s are virtually the same for both molecules (~200–400 nM). Furthermore, IPI-504 inhibits the secretion of immunoglobulin light chain from the RPMI-8226 multiple myeloma cells (EC50 ~300 nM). Importantly, IPI-504 is active in tumor xenograft models of multiple myeloma. The data indicate that active metabolites of IPI-504 accumulate in these xenografts long after these metabolites are cleared from the plasma compartment, suggesting that they preferentially accumulate in tumor cells based on their increased affinity to Hsp90 derived from tumor cells. In conclusion, we have developed IPI-504 as a novel, potent inhibitor of Hsp90 with greatly increased solubility over 17-AAG, and that IPI-504 is an active anti-tumor agent in vitro and in vivo.

Download Full-text

Biochemical Basis for Interactions Between Thalidomide and Inhibitors of the Isoprenoid Biosynthetic Pathway in Multiple Myeloma Cells

Blood ◽

10.1182/blood.v112.11.2635.2635 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2635-2635

Author(s):

Sarah A. Holstein ◽

Huaxiang Tong ◽

Raymond J. Hohl

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Annexin V ◽

Differential Sensitivity ◽

Parp Cleavage ◽

Myeloma Cells ◽

Protein Isoprenylation ◽

Geranylgeranyl Transferase ◽

Synthase Inhibitor ◽

Rpmi 8226

Abstract Introduction: The isoprenoid biosynthetic pathway (IBP) is responsible for the production of key sterol and nonsterol species, including farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) which serve as substrates for protein isoprenylation reactions. Several agents known to target the IBP have been observed to have cytotoxic effects in multiple myeloma cells. Thalidomide (Thal) has emerged as an effective agent for treating multiple myeloma. While Thal has been noted to have a variety of direct and indirect effects on myeloma cells, the precise mechanism of action remains unknown. Aim: We examined interactions between inhibitors of the IBP and Thal in multiple myeloma cells. The mechanisms underlying the observed differential sensitivity to these agents were explored. Methods: Studies were performed in three human multiple myeloma cell lines (RPMI-8226, U266, H929). Cytotoxicity was assessed via MTT assays, while apoptosis induction was determined by Annexin V staining and evaluation of PARP cleavage. Western blot analysis was used to evaluate inhibition of protein isoprenylation. Intracellular FPP and GGPP levels were measured via enzymatic coupling to fluorescently-tagged peptides, HPLC fractionation and fluorescence detection. Pharmacologic manipulation of the IBP was achieved with the following agents: lovastatin (Lov) as an HMG-CoA reductase inhibitor, zoledronic acid (ZA) as a FPP synthase inhibitor, digeranyl bisphosphonate (DGBP) as a GGPP synthase inhibitor, FTI-277 as a farnesyl transferase inhibitor (FTI), and GGTI-286 as a geranylgeranyl transferase I inhibitor (GGTI). Results: Addition of Thal to Lov (at both 24 & 48h), zoledronic acid (at 48h), or DGBP (at 24 & 48h) in RPMI-8266 cells results in marked enhancement in cytotoxicity. Isobologram analysis could not be performed as Thal by itself does induce cytotoxicity in MTT assays. Although Lov induces cytotoxicity in a concentration- and time-dependent manner in the U266 and H929 cells, the addition of Thal did not result in increased cytotoxicity. Neither ZA nor DGBP induced cytotoxicity in the U266 cell line, while the H929 cell line showed effects only at 48 hours. Addition of Thal to FTI or GGTI did not result in enhanced cytotoxicity in tested cell lines. Annexin V experiments confirmed enhanced induction of apoptosis in RPMI-8226 cells incubated with the combination of Thal/Lov or Thal/DGBP. Add-back experiments revealed that the enhanced cytotoxicity/induction of apoptosis observed with the addition of Thal could be prevented with the addition of mevalonate or GGPP in Lov-treated cells or GGPP in DGBP-treated cells. PARP cleavage was demonstrated in RPMI-8226 and H929 cells treated with Lov or DGBP (with or without Thal) and in U266 cells treated with Lov. As expected, Lov resulted in the accumulation of unmodified forms of proteins normally farnesylated (Ras) and geranylgeranylated (Rap1a and Rab6) in these cells. Interestingly however, while DGBP led to accumulation of unmodified Rap1a and Rab6 in RPMI-8226 and H929 cells, no effect was seen in the U266 line. Examination of intracellular levels of FPP and GGPP revealed that the U266 line has markedly larger pools of FPP (8.5-fold) and GGPP (2.7-fold) compared to RPMI-8226 and that treatment with DGBP only partially depletes U266 cells of GGPP. Conclusions: These studies demonstrate an interaction between thalidomide and IBP inhibitors in multiple myeloma cells. These effects appear dependent on depletion of GGPP. Since treatment with a geranylgeranyl transferase-I inhibitor does not produce similar results, this suggests that substrates of geranylgeranyl transferase-II, such as the Rab proteins, may play critical roles in myeloma pathophysiology. The finding that intracellular levels of FPP and GGPP vary markedly amongst cell lines explains differential sensitivity of these cells to pharmacologic manipulation of the IBP and may also influence sensitivity to chemotherapeutic agents. Further studies will determine the extent to which isoprenoid pool sizes vary in primary samples and may ultimately allow for the identification of multiple myeloma patients who would benefit from the addition of an IBP inhibitor to their treatment plan. Figure Figure

Download Full-text

The Translation Initiation Factor eIF4E Is Overexpressed in Multiple Myeloma and Is Critical for Myeloma Cell Proliferation and Survival

Blood ◽

10.1182/blood.v118.21.1853.1853 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1853-1853 ◽

Cited By ~ 1

Author(s):

Shirong Li ◽

MeiHua Jin ◽

Ailing Liu ◽

Markus Y. Mapara ◽

Suzanne Lentzsch

Keyword(s):

Multiple Myeloma ◽

Protein Synthesis ◽

Translation Initiation ◽

Clinical Care ◽

Protein Translation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Mrna Levels ◽

Myeloma Cells ◽

Rpmi 8226

Abstract Abstract 1853 Methods: The translation initiation factor eIF4E is central to protein synthesis in general, and overexpression and/or activation of eIF4E is associated with a malignant phenotype by regulating oncogenic protein translation. Several previous publications indicate that aberrant control of protein synthesis contributes to lymphoma genesis but the exact role of protein translation in multiple myeloma (MM) is less clear. Therefore, understanding the mechanisms that control protein synthesis is an emerging new research area in MM with significant potential for developing innovative therapies. The goal of this study was to determine the role and regulation of eIF4E, as well as the effects of protein translation controlling drugs in MM. Results: By western blot analysis as well as RT-PCR we found that eIF4E protein and mRNA levels are significantly elevated (up to 20 fold) in MM cell lines (H929, RPMI-8226, MM.1S and OPM2) and primary myeloma cells compared to normal plasma cells. Silencing of eIF4E gene expression in RPMI-8226 MM cells by a stable and inducible shRNA system significantly decreased viability of myeloma cells (by ∼ 43%) but not of HEK 293 suggesting a higher dependency of MM cells to protein translation. Next we evaluated different drugs including pomalidomide, rapamycin, pp242, 4EGI-1 and ribavirin, that are known to inhibit protein synthesis for their effects on protein translation in MM. By m7GTP pull down assays we evaluated the effects of the different drugs on eIF4E expression and activity. Rapamycin blocked the phosphorylation of 4EBP1 and eIF4E release, and subsequently inhibited eIF4G binding. The compound 4EGI-1 decreased the interaction between eIF4E and eIF4G. Pomalidomide decreased eIF4E protein expression. All drugs inhibited MM cell DNA synthesis measured by 3H-Thymidine incorporation. Treatment with pomalidomide (10uM), rapamycin (40nM), pp242 (10uM), 4EGI1 (50uM) or ribavirin (50uM) for 48h significantly decreased (p<0.05) proliferation by 43–62% indicating that drugs controlling protein translation inhibit MM growth. We also found that all drugs decreased expression of eIF4E dependent targets such as cyclin D1 and c-myc. Conclusion: Here we show that eIF4E, a key player in translational control, is highly expressed in MM cells and critical for MM growth and survival. Therefore our study helps to understand the function and regulatory mechanism of eIF4E in MM. Further the evaluation of drugs targeting protein translation provides the basis for the optimization of current MM treatment or to open up new strategies such as targeting protein translation in future MM therapy. Disclosures: Lentzsch: Celgene Corp: Consultancy, Research Funding; Onyx: Consultancy; Genzyme: Consultancy; prIME Oncology: Honoraria; Imedex: Honoraria; Clinical Care Options: Honoraria.

Download Full-text

Dual Antitumoral and Bone Antiresorptive Effect Of The Pan-Pim Kinase Inhibitor, LGH447, In Multiple Myeloma

Blood ◽

10.1182/blood.v122.21.4435.4435 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4435-4435

Author(s):

Teresa Paíno ◽

Antonio Garcia-Gomez ◽

Lorena González-Méndez ◽

Laura San-Segundo ◽

Montserrat Martín-Sánchez ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Kinase Inhibitor ◽

Annexin V ◽

Myeloma Cells ◽

Pim Kinases ◽

Castilla Y Leon ◽

Ic50 Values ◽

Rpmi 8226 ◽

Resorptive Activity

Introduction Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells in the bone marrow (BM) and is closely associated with osteolytic lesions, in part due to an increase in the bone-resorptive activity and number of osteoclasts (OCs). The activation of survival pathways in myeloma cells could be the cause of treatment failure rendering the disease incurable. Pim kinases are a family of survival serine/threonine kinases composed of three members (Pim1, Pim2 and Pim3) that are overexpressed in MM cells and may have a role in MM pathogenesis. However, little is known about the role of Pim kinases in OCs and its involvement in myeloma bone disease. Here, we have evaluated the preclinical activity of a new pan-Pim kinase inhibitor, LGH447, on MM cells and OCs. Cell lines, primary samples, material and methods LGH447 was provided by Novartis Pharmaceuticals. The human MM cell lines MM1S, MM1R, RPMI-8226 (or RPMI-8226-luc), RPMI-LR5, MM144, NCI-H929, OPM-2, U266, U266-Dox4 and U266-LR7 were employed. PBMCs from healthy volunteers were used to generate OCs, whereas primary mesenchymal stromal cells (MSCs) were obtained from bone marrow aspirates of MM patients. Cell viability was studied using MTT colorimetric assay or bioluminescence. Apoptosis was measured by annexin-V staining. For cell cycle analysis, propidium iodide staining was used. OC formation was assessed by enumeration of multinucleated (≥3) TRAP-positive cells and OC resorption was assessed on calcium-coated slides. Immunoblotting, quantitative PCR and immunofluorescence were used to further investigate the mechanism of action of LGH447. Results All MM cell lines expressed the three isoforms of Pim kinases with higher levels of Pim2. The dose-response curves to LGH447 after a 48 hour treatment revealed two groups of MM cell lines with regard to sensitivity to this drug: high sensitive, with IC50 values ranging from 0.2 to 3.3 µM (MM1S, MM1R, RPMI-8226, MM144, U266 and NCI-H929); and low sensitive, with IC50 values >7 µM (OPM-2, RPMI-LR5, U266-Dox4 and U266-LR7). Our results indicated that LGH447 promoted apoptosis in myeloma cells as shown by the increase in annexin-V positive cells and by the cleavage of initiator (caspases 8 and 9) and effector caspases (caspases 3 and 7) and of PARP. LGH447 also blocked the cell cycle in MM cells as demonstrated by the increase in G0-G1 and the decrease in S-G2-M phases. Importantly, LGH447 was also able to overcome the growth advantage conferred to RPMI-8226-luc cells by co-culture with MSCs or OCs. Regarding the mechanisms involved in these effects, LGH447 inhibited the mTOR pathway, demonstrated by a decreased phosphorylation of the downstream mTOR effectors, 4EBP1 and S6 in residues Thr37/46 and Ser235/236, respectively. Interestingly, LGH447 also inhibited OC formation and resorption activity. LGH447 treatment of human pre-OCs diminished the expression of key molecules involved in OC differentiation (p-Erk1/2 and NFATc1) and function [CAII (carbonic anhidrase II), CLCN7 (chloride channel 7), ATP6V1A (vacuolar-H+-ATPase catalytic subunit A1) and MMP9 (matrix metalloproteinase 9)] and also disrupted the F-actin ring necessary for OC effective resorption. Conclusion Overall, our results demonstrate that both MM cells and OCs are targets of the pan-Pim kinase inhibitor, LGH447. Therefore, the inhibition of Pim kinases could potentially provide a dual benefit in myeloma patients as a consequence of cytotoxic effects exerted on MM cells and an anti-resorptive activity on bone. This work was supported by funding from the Fundación Española de Hematología y Hemoterapia (AG-G), Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, the RTICC-Hematology Group (RD12/0036/0058), Spanish FIS (PI12/02591) and the Junta de Castilla y León, Gerencia Regional de Salud (GRS 862/A/13). Disclosures: Off Label Use: LGH447 is a pan-Pim kinase inhibitor (Novartis Pharmaceuticals). It has been used for pre-clinical studies in multiple myeloma.

Download Full-text

Induced Apoptosis Of Multidrug Resistance Leukemia K562/A02 Cells By Magnetic Nanoparticle Of Fe3O4 Copolymerizating JNK1 shRNA Expression Vector and Daunorubicin

Blood ◽

10.1182/blood.v122.21.4925.4925 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4925-4925

Author(s):

Wei Zhang ◽

Baoan Chen ◽

Xiangfeng He ◽

Rong Fu

Keyword(s):

Multidrug Resistance ◽

Protein Level ◽

Magnetic Nanoparticle ◽

Expression Vector ◽

Caspase 3 ◽

Chromatin Condensation ◽

Dapi Staining ◽

Shrna Expression ◽

Chemotherapeutic Regimen ◽

Shrna Expression Vector

Abstract Currently, serious toxicity and poor antitumor efficacy resulting from the induction of multidrug resistance (MDR) in hematological malignancies and solid tumors are common hindrances to successful systemic chemotherapy. One of the important mechanisms of tumorigenesis and resistance to anticancer drugs was blockade of the apoptosis-inducing pathway. In order to overcome tumor MDR while maintaining good antitumor activity, biomaterial combine with gene therapy provides a novel strategy for the treatment of MDR.The aim of this study was to explore the induction of apoptosis by magnetic nanoparticle of Fe3O4 (MNPs-Fe3O4), a novel delivery system for chemotherapeutic regimen that is chemically stable, environmentally friendly, and noncytotoxic, copolymerizating JNK1shRNA expression vector with DNR, which could overcome siRNA technique instability and enhance DNR chemotherapeutic efficiency and provide a new field to reverse tumor MDR. JNK1shRNA expression vector was constructed and screened the most effective plasmid and transfected the cells. Typical apoptotic characteristics and apoptosis effect of MNPs-Fe3O4 and PGM-3 with DNR were investigated by DAPI staining and FCM assay, respectively. Additionally, drug accumulation and apoptosis relative-genes were evaluated in K562/AO2 leukemia cells.The results showed that PGCsilencer-U6-neo-GFP-GV102 shRNA/JNK1 was successfully constructed, which was confirmed by sequencing that expression of JNK1 gene in mRNA and protein level that had the less in PGM-3 than that of PGM-1 or PGM-2. Meanwhile, MNPs-Fe3O4 and PGM-3 with DNR (DNR/PGM-3/MNPs-Fe3O4) could synergistically induced typical apoptotic characteristics of chromatin condensation and apoptotic body with DAPI staining, and a higher rate of apoptosis were detected in K562/A02 cells treated with DNR/PGM-3/MNPs-Fe3O4. Intracellular DNR was higher in K562/A02 cells treated with DNR/PGM-3/MNPs-Fe3O4 than that of DNR/MNPs-Fe3O4 by FCM analysis. Further study demonstrated that both DNR/PGM-3/MNPs-Fe3O4 and DNR/PGM-3 reduced the gene transcriptions and protein expressions of bcl-2, enhanced that of bax and caspase-3, decreased survivin in protein level, and has no different change in mRNA level by qRT-PCR and Western blotting analyses, respectively. These findings showed that the combination of MNPs-Fe3O4 with JNK1 shRNA expression vector and DNR could induce apoptosis of K562/A02 cells through elevating the ratio of bax/bcl-2, activating caspase-3, and decreasing survivin in protein level. Disclosures: No relevant conflicts of interest to declare.

Download Full-text