To the editor:
According to the World Health Organization classification, the natural killer (NK)–cell neoplasms consist of 2 separate entities: aggressive NK–cell leukemia (ANKL) and extranodal NK-cell lymphoma, nasal type (ENKL).1 Both of them are aggressive diseases with poor survival.2 We have previously reported constitutively active nuclear factor-κB signaling in ENKL.3 Here, we report that this earlier finding has given us a promising lead for NK-cell neoplasm treatment. Bortezomib, the proteasome inhibitor, targets nuclear factor-κB activation and can be used to treat multiple myeloma and mantle-cell lymphoma.4 We show in this study that bortezomib also has anticancer activity against ANKL and ENKL. Our results from both in vitro cytotoxicity assay and in vivo animal model give substantial support for planning a high priority clinical trial.
We used the cell viability assay to examine bortezomib's cytotoxicity on NK leukemia (KHYG-1, YT, and NK-92) and NK lymphoma (NK-YS) cell lines and short-term primary cultures from tumor biopsies of 2 patients with ENKL (Figure 1A).5 From the cytotoxicity results, we estimated that the median inhibitory concentrations (IC50) of bortezomib were 2.4 to 5 ng/mL in these neoplastic NK-cell lines and primary ENKL patient samples, approximately the same as in multiple myeloma.6 To further distinguish viable from nonviable cells, we stained the bortezomib-treated malignant NK cells with propidium iodide (PI) and found that they were permeable to PI and thus stainable by PI within 1 day of exposure (Figure 1B). Since the PI positivity is present only during late apoptosis, we speculated that these NK cells died very soon after exposure to the drug.
We next identified the starting point of apoptosis in these neoplastic NK cells. KHYG-1 cells were collected every 3 hours after exposure to bortezomib and stained with Annexin V and PI. A population of apoptotic cells with the Annexin V+/PI− phenotype was present in the KHYG-1 cells within 6 hours (Figure 1C). As the mitochondrial pathway is reported to be critical for bortezomib-induced apoptosis,7 we measured mitochondrial membrane potential (Δmψ) in all of the NK-cell lines. Each of them showed a mild decrease of Δmψ at 6 hours, becoming more evident at 9 hours (Figure 1D). It appeared that apoptosis began within 6 hours of exposure to bortezomib.
These in vitro assays show that bortezomib is effective in killing NK-cell neoplasms, but would the same be found in vivo in an animal model? We implanted YT cells into nude mice and injected the mice intraperitoneally with 1 mg/kg bortezomib, a safe and moderate dose used in the previous mouse studies of this drug.8,–10 We monitored the animals by recording their tumor growth and health conditions. Overall, the animals tolerated the drug toxicity well, and at this safe dose, bortezomib dramatically inhibited tumor growth (P = .001; Figure 1E).
In this study, we have shown bortezomib to be active against the NK-cell neoplasms ANKL and ENKL. With consistent apoptosis induction in 4 NK-cell lines and activity against 2 primary ENKL patient samples, bortezomib should be assessed for its activity against ANKL and ENKL in clinical trials of these diseases.
Authorship
Correspondence: Gopesh Srivastava, Department of Pathology, The University of Hong Kong, Queen Mary Hospital, Hong Kong; e-mail: [email protected]; or Raymond H. Liang, Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong; e-mail: [email protected].
This work was supported by a grant from the Research Grants Council of Hong Kong Special Administrative Region, P. R. China (HKU 7627/06M, G.S. and R.H.L.). We thank Millennium Pharmaceuticals (Cambridge, MA) for providing bortezomib for this study (supplier agreement no. 211837).
This study was approved by the Institutional Review Board of the University of Hong Kong (IRB Reference Number: UW 05–070 T/733), and the Committee on the Use of Live Animals in Teaching and Research, The Faculty of Medicine, The University of Hong Kong (CULATR 1098–05).
Conflict-of-interest disclosure: The authors declare no competing financial interests.
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