Induction of intrinsic apoptosis in leukaemia stem cells and in vivo zebrafish model by betulonic acid isolated from Walsura pinnata Hassk (Meliaceae)

Kok Hoong Leong, Mohamad Azrul Mahdzir, Mohd Fadzli Md Din, Khalijah Awang, Yuka Tanaka, Kasem Kulkeaw, Tohru Ishitani, Daisuke Sugiyama

Research output: Contribution to journalArticle

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Abstract

Background Leukaemia stem cells (LSC) have been associated with disease relapse and chemotherapy resistance. Betulonic acid (BA), a pentacyclic lupane-type triterpenoid, was reported to exhibit cytotoxicity toward various cancer cells and to be capable of inducing intrinsic apoptosis in solid tumours. However, the in vitro and in vivo apoptotic effects of BA against LSC remain unknown. Hypothesis/Purpose We aimed to determine whether BA isolated from bark of Walsura pinnata Hassk (Meliaceae) has pro-apoptotic effects on LSC in in vitro and in vivo models. Study design/Methods The population of high purity LSC was isolated from the Kasumi-1 cell line using magnetic sorting and characterised by flow cytometry. Cell viability was assessed using the MTS assay to examine dose- and time-dependent effects. The colony formation assay was performed in MethoCult® H4435 enriched media. Apoptosis was analysed using Annexin-V and propidium iodide staining, mitochondrial transmembrane potential was studied using JC-1 staining, and expression of apoptosis related genes (BAX, Bcl-2 and survivin) was evaluated by real time-polymerase chain reaction (RT-PCR). Caspase 3/7 and 9 activities were monitored through Promega Caspase-Glo® over a period of 24 h. The in vivo antileukaemia activity was evaluated using LSC xenotransplanted zebrafish, observed for DNA fragmentation from apoptosis by TUNEL assay. Results BA maintained its potency against the LSC population in comparison to parental Kasumi-1 cells (fold differences ≤ 1.94) over various treatment time points and significantly inhibited the formation of colonies by LSC. Apoptosis was triggered by BA through the upregulation of BAX and suppression of Bcl-2 and survivin genes with the loss of mitochondrial transmembrane potential, leading to the activation of caspase 9 followed by downstream caspase 3/7. BA was able to suppressed leukaemia formation and induced apoptosis in LSC xenotransplanted zebrafish. Conclusions The results demonstrate that BA inhibited the proliferative and colonogenic properties of LSC. BA induced apoptosis in LSC through the mitochondria pathway and was effective in the in vivo zebrafish model. Therefore, BA could be a lead compound for further development into a chemotherapy agent against LSC.

Original languageEnglish
Pages (from-to)11-21
Number of pages11
JournalPhytomedicine
Volume26
DOIs
Publication statusPublished - Mar 15 2017

Fingerprint

Meliaceae
Zebrafish
Leukemia
Stem Cells
Apoptosis
Caspase 7
bcl-2 Genes
Caspase 9
Caspase 3
Membrane Potentials
betulonic acid
Staining and Labeling
Drug Therapy
Propidium
Annexin A5
In Situ Nick-End Labeling
DNA Fragmentation
Caspases
Population

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Pharmacology
  • Pharmaceutical Science
  • Drug Discovery
  • Complementary and alternative medicine

Cite this

Induction of intrinsic apoptosis in leukaemia stem cells and in vivo zebrafish model by betulonic acid isolated from Walsura pinnata Hassk (Meliaceae). / Leong, Kok Hoong; Mahdzir, Mohamad Azrul; Din, Mohd Fadzli Md; Awang, Khalijah; Tanaka, Yuka; Kulkeaw, Kasem; Ishitani, Tohru; Sugiyama, Daisuke.

In: Phytomedicine, Vol. 26, 15.03.2017, p. 11-21.

Research output: Contribution to journalArticle

Leong, Kok Hoong ; Mahdzir, Mohamad Azrul ; Din, Mohd Fadzli Md ; Awang, Khalijah ; Tanaka, Yuka ; Kulkeaw, Kasem ; Ishitani, Tohru ; Sugiyama, Daisuke. / Induction of intrinsic apoptosis in leukaemia stem cells and in vivo zebrafish model by betulonic acid isolated from Walsura pinnata Hassk (Meliaceae). In: Phytomedicine. 2017 ; Vol. 26. pp. 11-21.
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abstract = "Background Leukaemia stem cells (LSC) have been associated with disease relapse and chemotherapy resistance. Betulonic acid (BA), a pentacyclic lupane-type triterpenoid, was reported to exhibit cytotoxicity toward various cancer cells and to be capable of inducing intrinsic apoptosis in solid tumours. However, the in vitro and in vivo apoptotic effects of BA against LSC remain unknown. Hypothesis/Purpose We aimed to determine whether BA isolated from bark of Walsura pinnata Hassk (Meliaceae) has pro-apoptotic effects on LSC in in vitro and in vivo models. Study design/Methods The population of high purity LSC was isolated from the Kasumi-1 cell line using magnetic sorting and characterised by flow cytometry. Cell viability was assessed using the MTS assay to examine dose- and time-dependent effects. The colony formation assay was performed in MethoCult{\circledR} H4435 enriched media. Apoptosis was analysed using Annexin-V and propidium iodide staining, mitochondrial transmembrane potential was studied using JC-1 staining, and expression of apoptosis related genes (BAX, Bcl-2 and survivin) was evaluated by real time-polymerase chain reaction (RT-PCR). Caspase 3/7 and 9 activities were monitored through Promega Caspase-Glo{\circledR} over a period of 24 h. The in vivo antileukaemia activity was evaluated using LSC xenotransplanted zebrafish, observed for DNA fragmentation from apoptosis by TUNEL assay. Results BA maintained its potency against the LSC population in comparison to parental Kasumi-1 cells (fold differences ≤ 1.94) over various treatment time points and significantly inhibited the formation of colonies by LSC. Apoptosis was triggered by BA through the upregulation of BAX and suppression of Bcl-2 and survivin genes with the loss of mitochondrial transmembrane potential, leading to the activation of caspase 9 followed by downstream caspase 3/7. BA was able to suppressed leukaemia formation and induced apoptosis in LSC xenotransplanted zebrafish. Conclusions The results demonstrate that BA inhibited the proliferative and colonogenic properties of LSC. BA induced apoptosis in LSC through the mitochondria pathway and was effective in the in vivo zebrafish model. Therefore, BA could be a lead compound for further development into a chemotherapy agent against LSC.",
author = "Leong, {Kok Hoong} and Mahdzir, {Mohamad Azrul} and Din, {Mohd Fadzli Md} and Khalijah Awang and Yuka Tanaka and Kasem Kulkeaw and Tohru Ishitani and Daisuke Sugiyama",
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T1 - Induction of intrinsic apoptosis in leukaemia stem cells and in vivo zebrafish model by betulonic acid isolated from Walsura pinnata Hassk (Meliaceae)

AU - Leong, Kok Hoong

AU - Mahdzir, Mohamad Azrul

AU - Din, Mohd Fadzli Md

AU - Awang, Khalijah

AU - Tanaka, Yuka

AU - Kulkeaw, Kasem

AU - Ishitani, Tohru

AU - Sugiyama, Daisuke

PY - 2017/3/15

Y1 - 2017/3/15

N2 - Background Leukaemia stem cells (LSC) have been associated with disease relapse and chemotherapy resistance. Betulonic acid (BA), a pentacyclic lupane-type triterpenoid, was reported to exhibit cytotoxicity toward various cancer cells and to be capable of inducing intrinsic apoptosis in solid tumours. However, the in vitro and in vivo apoptotic effects of BA against LSC remain unknown. Hypothesis/Purpose We aimed to determine whether BA isolated from bark of Walsura pinnata Hassk (Meliaceae) has pro-apoptotic effects on LSC in in vitro and in vivo models. Study design/Methods The population of high purity LSC was isolated from the Kasumi-1 cell line using magnetic sorting and characterised by flow cytometry. Cell viability was assessed using the MTS assay to examine dose- and time-dependent effects. The colony formation assay was performed in MethoCult® H4435 enriched media. Apoptosis was analysed using Annexin-V and propidium iodide staining, mitochondrial transmembrane potential was studied using JC-1 staining, and expression of apoptosis related genes (BAX, Bcl-2 and survivin) was evaluated by real time-polymerase chain reaction (RT-PCR). Caspase 3/7 and 9 activities were monitored through Promega Caspase-Glo® over a period of 24 h. The in vivo antileukaemia activity was evaluated using LSC xenotransplanted zebrafish, observed for DNA fragmentation from apoptosis by TUNEL assay. Results BA maintained its potency against the LSC population in comparison to parental Kasumi-1 cells (fold differences ≤ 1.94) over various treatment time points and significantly inhibited the formation of colonies by LSC. Apoptosis was triggered by BA through the upregulation of BAX and suppression of Bcl-2 and survivin genes with the loss of mitochondrial transmembrane potential, leading to the activation of caspase 9 followed by downstream caspase 3/7. BA was able to suppressed leukaemia formation and induced apoptosis in LSC xenotransplanted zebrafish. Conclusions The results demonstrate that BA inhibited the proliferative and colonogenic properties of LSC. BA induced apoptosis in LSC through the mitochondria pathway and was effective in the in vivo zebrafish model. Therefore, BA could be a lead compound for further development into a chemotherapy agent against LSC.

AB - Background Leukaemia stem cells (LSC) have been associated with disease relapse and chemotherapy resistance. Betulonic acid (BA), a pentacyclic lupane-type triterpenoid, was reported to exhibit cytotoxicity toward various cancer cells and to be capable of inducing intrinsic apoptosis in solid tumours. However, the in vitro and in vivo apoptotic effects of BA against LSC remain unknown. Hypothesis/Purpose We aimed to determine whether BA isolated from bark of Walsura pinnata Hassk (Meliaceae) has pro-apoptotic effects on LSC in in vitro and in vivo models. Study design/Methods The population of high purity LSC was isolated from the Kasumi-1 cell line using magnetic sorting and characterised by flow cytometry. Cell viability was assessed using the MTS assay to examine dose- and time-dependent effects. The colony formation assay was performed in MethoCult® H4435 enriched media. Apoptosis was analysed using Annexin-V and propidium iodide staining, mitochondrial transmembrane potential was studied using JC-1 staining, and expression of apoptosis related genes (BAX, Bcl-2 and survivin) was evaluated by real time-polymerase chain reaction (RT-PCR). Caspase 3/7 and 9 activities were monitored through Promega Caspase-Glo® over a period of 24 h. The in vivo antileukaemia activity was evaluated using LSC xenotransplanted zebrafish, observed for DNA fragmentation from apoptosis by TUNEL assay. Results BA maintained its potency against the LSC population in comparison to parental Kasumi-1 cells (fold differences ≤ 1.94) over various treatment time points and significantly inhibited the formation of colonies by LSC. Apoptosis was triggered by BA through the upregulation of BAX and suppression of Bcl-2 and survivin genes with the loss of mitochondrial transmembrane potential, leading to the activation of caspase 9 followed by downstream caspase 3/7. BA was able to suppressed leukaemia formation and induced apoptosis in LSC xenotransplanted zebrafish. Conclusions The results demonstrate that BA inhibited the proliferative and colonogenic properties of LSC. BA induced apoptosis in LSC through the mitochondria pathway and was effective in the in vivo zebrafish model. Therefore, BA could be a lead compound for further development into a chemotherapy agent against LSC.

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