Elsevier

Immunobiology

Volume 220, Issue 3, March 2015, Pages 331-340
Immunobiology

Decreased expression of nucleophosmin/B23 increases drug sensitivity of adriamycin-resistant Molt-4 leukemia cells through mdr-1 regulation and Akt/mTOR signaling

https://doi.org/10.1016/j.imbio.2014.10.015Get rights and content

Abstract

Nucleophosmin/B23 (NPM) is a nuclear protein with prosurvival and ribosomal RNA processing functions. However, the potential role of NPM involved in drug-resistance in leukemia has not been investigated clearly. In this study, we generated an adriamycin (ADM)-resistant lymphoblastic cell line Molt-4/ADR (MAR) by stepwise induction. Cell proliferation, sensitivity to chemotherapy agents and expressions of drug resistance related molecules were assessed. The IC50 of Molt-4 cells were 0.58 ± 0.11 μmol/L and MAR cells were 22.56 ± 1.94 μmol/L, meaning MAR cells were 38.63 fold resistant to Molt-4 cells. Furthermore, MAR cells gained an expression of mdr-1 (P-gp) and a higher expression of NPM compared to Molt-4 cells. Knockdown of NPM by RNA interference (RNAi) suppressed the viability of both Molt-4 and MAR cells. After NPM RNAi, the IC50 of MAR and Molt-4 cells were 3.83 ± 0.38 μmol/L and 0.19 ± 0.02 μmol/L respectively. Both of them revealed an increase of drug sensitivity with down-regulation of mdr-1 and Akt/mTOR signaling. Knockdown of mdr-1 could also reverse the drug resistance, with no change in NPM expression. It could be concluded that knockdown of NPM reversed the drug resistance by down-regulating P-gp and Akt/mTOR signal pathway, indicating that NPM may serve as a potential modulator in drug resistance.

Introduction

Adult acute lymphoblastic leukemia (ALL) is a heterogeneous hematological malignancy. There are two subtypes of ALL, B-lineage ALL (B-ALL) accounts for 85% of all cases and the remaining 15% are T-lineage ALL (T-ALL). Compared to children, who can approach a cure rate of 90%, a majority of adult patients failed to attain disease eradication, especially the prognosis in patients with relapsed or refractory disease is uniformly poor (Pui et al. 2008).

Many factors have been proved to be associated with ALL prognosis, such as immunologic subtype, and clinical, genetic, and molecular features (Lazarus and Advani 2012), several of which have clear implications for risk stratification and targeted therapeutic intervention (Mullighan 2012). T-ALL is known to have less favorable prognosis and relapse is more common probably due to emergence of chemo-resistance. The principal source of ALL relapse is the survival and expansion of leukemic cells resistant to chemotherapy. Researches over past three decades have identified a number of ways in which cancer cells can elude chemotherapy (Gottesman et al. 2002); including ATP-binding cassette (ABC) transporters mediated drug efflux and other mechanism.

Nucleophosmin (NPM, also known as B23, numatrin, or N038) is regularly identified as multifunctional nuclear protein (Maggi et al., 2008, Yu et al., 2006, Okuwaki, 2008, Mukudai et al., 2008), not only an important player in ribosome biogenesis but also a potential regulator for cell proliferation. Aberrant expression of NPM, such as mutation, deletion, over-expression or rearrangement, could lead to malignant transformation in tumor cells (Grisendi et al. 2006). It is reported that over-expression of NPM in solid tumor had been detected as a poor prognostic factor and related to drug-resistance development (Yang et al. 2007). While in adult de novo acute myeloid leukemia (AML), NPM1 mutations occurs in about 30% of patients, and approximately 85% of AML patients with NPM1 mutation show a normal karyotype subgroup (AML-NK) (Verhaak et al. 2005). Researchers found that mutated NPM without FLT3-ITD showed a good response to chemotherapy in AML, while patients carrying both NPM1/FLT3-ITD mutations predicted poor responses (Schnittger et al. 2005).

Although a great deal of knowledge of NPM has been accumulated, little information on the role of NPM in drug resistance in leukemia is available. According to our previous investigation, NPM levels are up regulated in drug-resistant cell lines and primary leukemic cells. It is revealed that an expression of NPM together with nucleolin/C23 (NCL), another important nuclear protein, has relationship to drug resistance. High expressions of NPM and NCL could be observed in refractory or relapsed acute leukemia patients, which significantly correlated with poor prognosis (Hu et al. 2011). Moreover, the expressions of both proteins were confirmed in 9 hematologic cell line, especially in drug resistant cell line HL60/ADR, K562/ADR and KG/01. It is confirmed that in resistant myeloid leukemia HL-60/ADR cells, knockdown of NPM could reverse multidrug resistance (Lin et al. 2013).

In this study, we wanted to evaluate whether NPM is involved in drug resistance in lymphocytic leukemia cells. Since the lymphocytic cell lines with multidrug resistance are not common in laboratory, we had to induce the drug resistant cell line by induction with increasing concentration of Adriamycin. We also investigated the potential involvement of NPM in cell drug resistance and explored the possibility of modulating drug sensitivity by knocking down NPM.

Section snippets

Cell culture and drug-resistance induction

Molt-4 cell line kept in our laboratory (Fujian Institute of Hematology, Fuzhou, China) was cultured in RPMI-1640 medium (Hyclone, UT, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone, UT, USA) at 37 °C in a 5% CO2 incubator (Thermo Fisher Scientific, Rockford, IL, USA). Adriamycin (ADM, Sigma–Aldrich, St. Louis, MO, USA) was dissolved in normal saline (NS) with a concentration of 2 mg/ml. To establish an adriamycin-resistant leukemia cell line, Molt-4 cells were exposed to stepwise

Molt-4/ADR (MAR) cell line showed extraordinary drug-resistance

ADM-resistant counterparts of Molt-4 were generated by culturing parental cells in the presence of increasing concentrations of ADM. After drug exposure for at least 6 months, MAR cells from a single clone could survive in a high concentration of ADM. IC50 of MAR cells were raised as high as 22.56 ± 1.94 μmol/L while its parental cell line Molt-4 only 0.58 ± 0.11 μmol/L, which is 38.63 times. IC50 value fluctuated slightly with ADM-exposure time. These cells were not only resistant to other MDR

Discussion

Durable leukemic cell eradication by chemotherapy is challenged by the development of multidrug-resistance (MDR). As experimental models, chemotherapy-resistant cell line can be generated by in vitro selection with cytotoxic agents, which can become resistant to a single drug or a class of drugs. After selection for resistance to ADM, MAR cells show cross-resistance to other structurally and mechanistically unrelated drugs.

Many studies were concentrated on the mechanisms of drug resistance.

Conclusion

It could be concluded that knockdown of NPM reversed the drug resistance by down-regulating drug efflux pump protein P-gp and Akt/mTOR signal pathway, indicating that NPM may serve as a potential modulator in drug resistance. Further investigation is needed to explore more precise mechanisms of NPM gene in drug-resistance in leukemia.

Conflict of interest

No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part. All the authors listed have approved the manuscript that is enclosed.

Acknowledgements

This work was supported by National Natural Science Foundation of China (81270608), National High Technology Research and Development Program of China-863 Program (2012AA02A505), National Public Health Grand Research Foundation (201202017), Fujian Provincial Key Scientific and Technological Projects (2011Y0026), Educational Commission of Fujian Province, China (JA12158), National Key Clinical Specialty Discipline Construction Program, China ([2011]1018), and Fujian Provicial Key Clinical

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    These authors contributed equally to this work.

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