Single Agent and Synergistic Activity of Maritoclax with ABT-263 in Nasopharyngeal Carcinoma (NPC) Cell Lines

Abstract

The BCL-2 anti-apoptotic proteins are over-expressed in many cancers and hence are attractive therapeutic targets. In this study, we tested the sensitivity of two Nasopharyngeal Carcinoma (NPC) cell lines HK1 and C666-1 to Maritoclax, which is reported to repress anti-apoptotic protein MCL-1 and BH3 mimetic ABT-263, which selectively inhibits anti-apoptotic proteins BCL-2, BCL-XL and BCL-w. We investigated the sensitisation of the NPC cell lines to these drugs using the SYBR Green I assay and 3D NPC spheroids. We report that Maritoclax repressed anti-apoptotic proteins MCL-1, BCL-2, and BCL-XL in a dose- and time-dependent manner and displayed a single agent activity in inhibiting cell proliferation of the NPC cell lines. Moreover, combination of Maritoclax and ABT-263 exhibited synergistic antiproliferative effect in the HK1 cells. Similar results were obtained in the 3D spheroids generated from the HK1 cells. More notably, 3D HK1 spheroids either treated with single agent Maritoclax or combination with ABT-263, over 10 days, did not develop resistance to the treatment rapidly. Collectively, the findings illustrate that Maritoclax as a single agent or combination with BH3 mimetics could be potentially useful as treatment strategies for the management of NPC.

Keywords: 3D NPC Spheroids; ABT-263; BH3 Mimetics; Maritoclax; Nasopharyngeal Carcinoma.

Figure 1

Maritoclax repressed the anti-apoptotic proteins in a dose- and time-dependent manner. (a) Basal expression levels of the anti-apoptotic proteins in the NPC cell lines. The basal expression levels of MCL-1, BCL-2 and BCL-XL in the C666-1 and the HK1 cells were determined by SDS-PAGE gel electrophoresis. NPC cell lines HK1 and C666-1 cells were treated with (b, d) Escalating doses of Maritoclax for 24 h to study the dose response or (c, e) with 2 μM or 1 μM of Maritoclax in the HK1 and C666-1 cells, respectively, at the indicated time points to study the time response. Immunoblot analyses illustrated that repression of MCL-1 by Maritoclax in both cell lines were dose- and time-dependent. (b–c) Maritoclax demonstrated a modest dose and time effect on the expression level of BCL-XL in the HK1 cells. (d–e) Maritoclax had a transient dose effect but a strong time effect on the level of BCL-2 in the C666-1 cells. Alpha-tubulin was used as the loading control. Arrows indicate the protein bands probed on the x-ray films.

Figure 2

Maritoclax exhibited single agent activity in inhibiting cell proliferation of the NPC cells. (a) HK1 cells and (b) C666-1 cells were treated with increasing concentrations of ABT-263 (0–32 μM) (open circle) or Maritoclax (0–32 μM) (open diamond) or combination of ABT-263 and Maritoclax (open square) at 1:1 drug concentration ratio for 72 h. Cell proliferation was assessed using the SyBr Green I assay. Points represent mean ± SEM of four experiments. (c) One representative experiment showing HK1 spheroids demonstrating decrease viable cells (calcein-AM) and increase dead cells (Ethidium homodimer I) after treatment with single agent Maritoclax at increasing concentrations over 10 days. Medium and drug were replenished every 72 h. Size bar: 500 μM.

Figure 3

Synergistic anti-proliferative effects of ABT-263 and Maritoclax in the HK1 cells. (a) HK1 cells were treated with increasing concentrations of ABT-263 (0–32 μM) in the presence or absence of either 0.5 or 1 μM of Maritoclax for 72 h. Cell proliferation was assessed using the SyBr Green I assay. Points represent mean ± SEM of four experiments. (b) One representative experiment showing HK1 spheroids demonstrating decrease viable cells (calcein-AM) and increase dead cells (Ethidium homodimer I) after treatment with single agent Maritoclax and ABT-263 and combination of both drugs at increasing concentrations over 10 days. Size bar: 500 μM.