Knockdown of Bcl-xL enhances growth-inhibiting and apoptosis-inducing effects of resveratrol and clofarabine in malignant mesothelioma H-2452 cells

Our plain-English summary of Knockdown of Bcl-xL enhances growth-inhibiting and apoptosis-inducing effects of resveratrol and clofarabine in malignant mesothelioma H-2452 cells, by Y.J. Lee, I.S. Hwang, Y.J. Lee, et al (Journal of Korean Medical Science, 2014).

The study

• Authors: Y.J. Lee, I.S. Hwang, Y.J. Lee, C.H. Lee, S.H. Kim, H.S. Nam, Y.J. Choi, S.H. Lee
• Journal: Journal of Korean Medical Science
• Published: 2014;29(11):1464-72
• Cited by: 10 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Mcl-1 and Bcl-xL, key anti-apoptotic proteins of the Bcl-2 family, have attracted attention as important molecules in the cell survival and drug resistance. In this study, we investigated whether inhibition of Bcl-xL influences cell growth and apoptosis against simultaneous treatment of resveratrol and clofarabine in the human malignant mesothelioma H-2452 cells. Resveratrol and clofarabine decreased Mcl-1 protein levels but had little effect on Bcl-xL levels. In the presence of two compounds, any detectable change in the Mcl-1 mRNA levels was not observed in RT-PCR analysis, whereas pretreatment with the proteasome inhibitor MG132 led to its accumulation to levels far above basal levels. The knockdown of Bcl-xL inhibited cell proliferation with cell accumulation at G2/M phase and the appearance of sub-G0/G1 peak in DNA flow cytometric assay. The suppression of cell growth was accompanied by an increase in the caspase-3/7 activity with the resultant cleavages of procaspase-3 and its substrate poly (ADP-ribose) polymerase, and increased percentage of apoptotic propensities in annexin V binding assay. Collectively, our data represent that the efficacy of resveratrol and clofarabine for apoptosis induction was substantially enhanced by Bcl-xL-lowering strategy in which the simultaneous targeting of Mcl-1 and Bcl-xL could be a more effective strategy for treating malignant mesothelioma.

In plain English

This was a laboratory study of cultured cells, not people or animals, and it did not involve asbestos exposure. The authors worked with H-2452, a human malignant mesothelioma cell line, which they described as only mildly sensitive to a combination of resveratrol (15 μM) and the chemotherapy drug clofarabine (40 nM). After 72 hours, this combination reduced cell growth by approximately 29.6%. In their earlier experiments with another mesothelioma cell line, MSTO-211H, the reduction was approximately 55%. The combination lowered the amount of Mcl-1, a protein that helps cells avoid programmed cell death (apoptosis), without changing Mcl-1 messenger RNA. Levels of a related protein, Bcl-xL, did not change significantly. The authors then used small interfering RNA to reduce Bcl-xL, alone or together with Mcl-1. This slowed cell growth, and the effect was greater when the two drugs were added. Markers of apoptosis increased, as did the share of cells stopped at the G2/M stage of division. The authors concluded that targeting Mcl-1 and Bcl-xL together inhibited growth and promoted apoptosis in these cells. They stated that studies in animal models are needed.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) cell-culture experiment using one human malignant mesothelioma cell line
• Who was studied: Cultured human malignant mesothelioma cells of the H-2452 line, obtained from the American Type Culture Collection. No human subjects or animals were studied.
• Exposure: No asbestos exposure was studied. Cells were treated with resveratrol (15 μM) plus clofarabine (40 nM), with or without prior small interfering RNA knockdown of Bcl-xL (10 nM), alone or combined with Mcl-1 knockdown (10 nM).
• Main results:
  • In H-2452 cells, resveratrol up to 15 μM was non-toxic (IC10 = 17 μM). The next higher dose, 20 μM, significantly inhibited cell growth.
  • Over 72 hours, clofarabine alone at 20, 40, 80, 160 and 320 nM reduced H-2452 cell proliferation by approximately 9.9%, 12.8%, 17.2%, 24.8% and 40.5%.
  • Combined with resveratrol (15 μM), the same clofarabine concentrations reduced H-2452 cell proliferation by approximately 19.6%, 29.6%, 32.5%, 39.1% and 48.9%.
  • At 72 hours, resveratrol (15 μM) plus clofarabine (40 nM) inhibited H-2452 cell proliferation by approximately 29.6%. In the authors' previous experiments with MSTO-211H cells, approximately 55% was inhibited.
  • The combination treatment lowered Mcl-1 protein levels in H-2452 cells, and the levels stayed low over 72 hours.
  • The drop in Mcl-1 protein occurred without any change in Mcl-1 mRNA level.
  • The combination treatment caused no significant change in Bcl-xL protein level.
  • When protein production was blocked, Mcl-1 had a half-life of about 16.5 minutes with cycloheximide alone. With the proteasome inhibitor MG132 added, the half-life became longer than 2 hours.
  • Knockdown of Bcl-xL gradually inhibited H-2452 cell proliferation over 72 hours, and this was augmented by subsequent resveratrol plus clofarabine.
  • The sub-G0 peak, which the authors took as a sign of apoptosis, increased significantly after Bcl-xL knockdown.
  • The proportion of cells in the G2/M phase tended to increase in the order Mcl-1 plus Bcl-xL knockdown, then Bcl-xL knockdown, then control siRNA. The authors described this as cell cycle arrest.
  • Compared with control siRNA, Bcl-xL knockdown increased caspase-3/7 activity and enhanced cleavage of procaspase-3 and PARP.
  • The percentage of apoptotic cells measured by annexin V binding increased, and these findings became apparent after exposure to resveratrol plus clofarabine.
• The authors' conclusion: The authors concluded that targeting Mcl-1 and Bcl-xL at the same time inhibited cell proliferation, promoted apoptosis and blocked cell cycle progression in H-2452 mesothelioma cells. These were cells they described as mildly sensitive to resveratrol plus clofarabine.
• Limitations the authors state:
  • The authors stated that their study did not provide direct evidence that apoptosis was occurring in the cells arrested in the G2/M phase.
  • The authors stated that studies in animal models are required to determine whether this strategy may have potential in humans.

Topics

Medical terms in this study, explained in our Lexicon: Apoptosis, Mesothelioma.

Related studies

• Resveratrol and clofarabine induces a preferential apoptosis-activating effect on malignant mesothelioma cells by Mcl-1 down-regulation and caspase-3 activation — Y.J. Lee, Y.J. Lee, et al. (2015)
• Synergistic anti-cancer effects of resveratrol and chemotherapeutic agent clofarabine against human malignant mesothelioma MSTO-211H cells — Y.J. Lee, Y.J. Lee, et al. (2013)
• Synergistic inhibition of mesothelioma cell growth by the combination of clofarabine and resveratrol involves Nrf2 downregulation — Y.J. Lee, J.H. Im, et al. (2012)
• The flavonoid resveratrol suppresses growth of human malignant pleural mesothelioma cells through direct inhibition of specificity protein 1 — K.A. Lee, Y.J. Lee, et al. (2012)
• Resveratrol contributes to chemosensitivity of malignant mesothelioma cells with activation of p53 — Y.J. Lee, I.S. Park, et al. (2014)
• Cisplatin and resveratrol induce apoptosis and autophagy following oxidative stress in malignant mesothelioma cells — Y.J. Lee, G.J. Lee, et al. (2016)

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