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

Tien

et al. 2021

Cells

Targeting the activities of endoplasmic reticulum (ER)–mitochondrial-dependent metabolic reprogramming is considered one of the most promising strategies for cancer treatment. Here, we present biochemical subcellular fractionation, coimmunoprecipitation, gene manipulation, and pharmacologic evidence that induction of mitochondria-localized phospho (p)-cyclin dependent kinase 1 (CDK1) (Thr 161)–cyclin B1 complexes by apigenin in nasopharyngeal carcinoma (NPC) cells impairs the ER–mitochondrial bioenergetics and redox regulation of calcium (Ca++) homeostasis through suppressing the B cell lymphoma 2 (BCL-2)/BCL-2/B-cell lymphoma-extra large (BCL-xL)-modulated anti-apoptotic and metabolic functions. Using a specific inducer, inhibitor, or short hairpin RNA for acid sphingomyelinase (ASM) demonstrated that enhanced lipid raft-associated ASM activity confers alteration of the lipid composition of lipid raft membranes, which leads to perturbation of protein trafficking, and induces formation of p110α free p85α–unphosphorylated phosphatase and tensin homolog deleted from chromosome 10 complexes in the lipid raft membranes, causing disruption of phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-GTP-ras-related C3 botulinum toxin substrate 1 (Rac1)-mediated signaling, thus triggering the p-CDK1 (Thr 161))–cyclin B1-mediated BCL-2 (Thr 69/Ser 87)/BCL-xL (Ser 62) phosphorylation and accompanying impairment of ER–mitochondria-regulated bioenergetic, redox, and Ca++ homeostasis. Inhibition of apigenin-induced reactive oxygen species (ROS) generation by a ROS scavenger N-acetyl-L-cysteine blocked the lipid raft membrane localization and activation of ASM and formation of ceramide-enriched lipid raft membranes, returned PI3K-Akt-GTP-Rac1-modulated CDK1–cyclin B1 activity, and subsequently restored the BCL-2/BCL-xL-regulated ER–mitochondrial bioenergetic activity. Thus, this study reveals a novel molecular mechanism of the pro-apoptotic activity of ASM controlled by oxidative stress to modulate the ER–mitochondrial bioenergetic metabolism, as well as suggests the disruption of CDK1–cyclin B1-mediated BCL-2/BCL-xL oncogenic activity by triggering oxidative stress-ASM-induced PI3K-Akt-GTP-Rac1 inactivation as a therapeutic approach for NPC.

Section: Discussionmentioning

confidence: 99%

Oxidative Stress-Induced Unscheduled CDK1–Cyclin B1 Activity Impairs ER–Mitochondria-Mediated Bioenergetic Metabolism

Tien

et al. 2021

Cells

“…However, the role of BCL-XL for NPC cell survival was not interrogated in the same study. Our own study unveiled that co-inhibition of BCL-2 and BCL-XL did not induce cell killing in NPC [10]. This led to the re-evaluation of the utility of BCL-2 and BCL-XL as therapeutic targets in NPC.…”

Section: Introductionmentioning

confidence: 87%

“…Patients with negative BCL-2 expression were reported to display better disease free 5-year survival compared to patients with BCL-2 positive tumours [7]. There were number of studies which attempted to target the anti-apoptotic proteins for NPC treatment but the study ndings were unsatisfactory [8][9][10]. The problem lied largely on the failure to determine which anti-apoptotic proteins did NPC cells relied for survival.…”

Section: Introductionmentioning

confidence: 99%

Dual Inhibition of Anti-apoptotic Proteins BCL-XL and MCL-1 Enhances Cytotoxicity of Nasopharyngeal Carcinoma Cells

Rahman

Azlan

et al. 2021

Preprint

Self Cite

One of the many strategies that cancer cells evade death is through up-regulation of the BCL-2 anti-apoptotic proteins. Hence, these proteins have become attractive therapeutic targets. Given that different cell population rely on different anti-apoptotic proteins for survival, it is crucial to determine which proteins are important for Nasopharyngeal carcinoma (NPC) cell survival. Here we determined the survival requirements for the NPC cells using combination of the CRISPR/Cas9 technique and selective BH3-mimetics. A human apoptosis RT2 Profiler PCR Array was first employed to profile the anti-apoptotic gene expressions in NPC cell lines HK-1 and C666-1. The HK-1 cells expressed all the anti-apoptotic genes (MCL-1, BFL-1, BCL-2, BCL-XL, and BCL-w). Similarly, the C666-1 cells expressed all the anti-apoptotic genes except BFL-1 (undetectable level). Notably, both cell lines highly expressed MCL-1. Deletion of MCL-1 sensitized the NPC cells to BCL-XL selective inhibitor A-1331852, suggesting that MCL-1 and BCL-XL may be important for NPC cell survival. Co-inhibition of MCL-1 and BCL-2 with MCL-1 selective inhibitor S63845 and BCL-2 selective inhibitor ABT-199 inhibited NPC cell proliferation but the effect on cell viability was more profound with co-inhibition of MCL-1 and BCL-XL with S63845 and A-1331852, implying that MCL-1 and BCL-XL are crucial for NPC cell survival. Furthermore, co-inhibition of MCL-1 and BCL-XL inhibited the growth and invasion of NPC spheroids. Deletion of BFL-1 sensitized NPC cells to A-1331852 suggesting that BFL-1 may play a role in NPC cell survival. Taken together co-inhibition of BCL-XL and MCL-1/BFL-1 could be potential treatment strategies for NPC.

“…The Dynamic BH3 profiling [3], the CRISPR/Cas9 genome editing technique [4] and BH3 mimetics given their selectivity in inhibiting the antiapoptotic proteins (an approach that is known as 'chemical parsing') [5] are approaches that can be used to parse the individual contributions of the BCL-2 anti-apoptotic proteins for cell survival. Our previous study showed that ABT-263, a small molecule inhibitor which selectively inhibits BCL-2, BCL-XL and BCL-w, had minimal effect on NPC cells, which made it clear that other anti-apoptotic proteins may be important for NPC cell survival [6].…”

Section: Introductionmentioning

confidence: 99%

Dual inhibition of anti-apoptotic proteins BCL-XL and MCL-1 enhances cytotoxicity of Nasopharyngeal carcinoma cells

Rahman

Azlan

et al. 2021

Preprint

Self Cite

One of the many strategies that cancer cells use to evade cell death is through upregulation of the BCL-2 anti-apoptotic proteins. Hence, these proteins have become attractive therapeutic targets. Given that different cell population rely on different anti-apoptotic proteins for survival, it is crucial to determine which proteins are important for NPC survival. Here we determined the survival requirements for the NPC cells using combination of CRISPR/Cas9 technique and pharmacological approaches. A human apoptosis RT2 Profiler PCR Array was first employed to profile the anti-apoptotic gene expressions in NPC cell lines HK-1 and C666-1. The HK-1 cells expressed all the anti-apoptotic genes (MCL-1, BFL-1, BCL-2, BCL-XL, and BCL-w). Similarly, the C666-1 cells expressed all the anti-apoptotic proteins except BFL-1 (undetectable level). Notably, both cell lines highly expressed MCL-1. Deletion of MCL-1 sensitized cells to A-1331852 suggesting that MCL-1 and BCL-XL may be important for NPC cell survival. Co-inhibition of MCL-1 and BCL-2 with MCL-1 selective inhibitor S63845 and BCL-2 selective inhibitor ABT-199 inhibited NPC cell proliferation but the effect on cell viability was more profound with co-inhibition of MCL-1 and BCL-XL with S63845 and A-1331852, implying that MCL-1 and BCL-XL are crucial for NPC cell survival. Furthermore, co-inhibition of MCL-1 and BCL-XL inhibited the growth and invasion of NPC spheroids. Deletion of BFL-1 sensitized cells to A-1331852 suggesting that BFL-1 may play a role in NPC cell survival. Taken together co-inhibition of BCL-XL and MCL-1/BFL-1 could be potential treatment strategies for NPC.