Phytopharmaceuticals, biologically active compounds derived from plant origins, provide a diverse array of potential therapeutic agents 1 . Celastrol, a pentacyclic triterpene extracted from the species Tripterygium wilfordii , has demonstrated anti-inflammatory and antioxidant properties in vitro and exhibited anti-cancer properties against diverse cancer types 2 . This collaborative study with Kidscan Children’s Cancer Research and funded by a Biochemical Society Summer Research Studentship focuses on evaluating celastrol against medulloblastoma, emphasizing its efficacy against various subtypes and its potential for inducing cardiotoxicity.
The research unveils celastrol's potent cytotoxicity against medulloblastoma cells, particularly the group 3 medulloblastoma cell line HDMB03. Celastrol demonstrates effectiveness with EC 50 values (The concentration required to decrease viability by 50%) below 0.5μM (Figure 1A), through the induction of apoptotic cell death. Subsequent investigations extended to the Sonic Hedgehog medulloblastoma subtype, DAOY (Figure 1B), with similar findings observed validating celastrol's efficacy across different medulloblastoma subtypes.
The observed irreversible cytotoxicity after 24 hours of treatment with concentrations exceeding 500nM (Figures 1C-D), and fluorescent microscopy visualising the activation of Caspases 3 and 7, provide supporting evidence of irreversible caspase-mediated apoptotic cell death induced by celastrol in numerous medulloblastoma subtypes (Figure 2). These data position celastrol as a promising chemotherapeutic agent for medulloblastoma treatment.
As a part of this project, we also sought to preliminarily investigate the potential side effects of celastrol by evaluating its toxicity towards cardiac cells. This analysis explored the cardiotoxic potential of celastrol using an in vitro model of cardiotoxicity utilising H9c2 cardiac myoblasts. Results of these investigations indicate that concentrations greater than 2μM induce cardiotoxicity after 72 hours of treatment (Figure 3) highlighting some selectivity towards medulloblastoma cells but also highlighting potential cardiotoxicity at higher concentrations. This underscores the need for meticulous assessment of potential side effects of novel compounds early in the drug development pipeline.
Beyond its potential clinical application, the project encouraged scientific exploration of compounds derived from traditional medicine for therapeutic potential, an underexploited source of novel therapeutic compounds, and began to develop an understanding of their molecular mechanisms of action. The development of an in vitro model of cardiotoxicity for simultaneous screening of therapeutic and cardiotoxic potential holds promise for revolutionizing drug development, especially considering the relatively fewer off-target effects associated with phytochemicals compared to synthetic compounds.
The project has equipped me with a diverse set of practical and transferable skills crucial for embarking on a research career. From fundamental laboratory techniques to advanced methodologies like time-lapse live-cell microscopy and flow cytometry, I have gained proficiency and resilience in problem-solving. Workshops and presentations have further contributed to the development of my analytical and communication skills, priming me for postgraduate research opportunities or industrial research positions.
In conclusion, celastrol emerges as a promising and comparatively safe phytopharmaceutical candidate for medulloblastoma treatment. Ongoing research aims to delve into the molecular mechanisms of action and explore nano-encapsulation strategies to enhance potency and minimize off-target effects. The findings underscore the broader applicability of in vitro models for simultaneous screening of therapeutic and cardiotoxic potential, paving the way for the development of novel compounds in the realm of cancer therapeutics.
The author would like to thank the Biochemical Society for awarding them with the Biochemical Society Summer Studentship which enabled this research to be conducted and for exposure to be gained in academic research. Additionally, the author extends appreciation to Dr. Matthew Jones for endorsing their application for the studentship. Through his support, the author delivered an oral presentation summarizing the findings and conclusions to the research laboratory team and a broader academic audience. His guidance provided the author with valuable insights into the daily responsibilities of a research scientist involved in interdisciplinary research, an experience made possible by this studentship opportunity.
Figure 1 - The cytotoxic effects of celastrol against medulloblastoma subtypes. (A & B) Dose-response curves comparing the cytotoxicity of celastrol and against HD-MB03 (Panel A) and DAOY (Panel B) cell lines following treatment for 24 (Red), 48 (Blue) and 72 hours (Black). (C) Representative crystal violet staining images showing HD-MB03 colonies arising after celastrol wash-out over 10 days of recovery. Pre-treatments with celastrol were carried out over 48 hours before wash-out. (D) Corresponding Mean ± SD crystal violet absorbance derived from 6 independent repeats. All data compared to vehicle alone (0.5 % v/v DMSO). Statistical significance (P < 0.05) was determined following a one-way ANOVA with a Tukey’s post-hoc test **P < 0.01; ***P < 0.001; **** P < 0.0001. n = 6 independent repeats for all experiments.
Figure 2- The effects of celastrol on caspase-mediated apoptosis. Specimen light and fluorescent micrographs (100x total magnification) showing the activation of Caspases 3/7 following staining with Invitrogen Cell Event Caspase-3/7 detection agent following treatment with Celastrol (0.25, 0.5 and 1 µM) for 24 hours. Scale bars = 200 µm.
Figure 3 – The cardiotoxic effects of celastrol. Dose-response curves comparing the cytotoxicity of celastrol against H9c2 terminally differentiated cardiac myocytes following treatment for 72 hours. Statistical significance (P < 0.05) was determined following a one-way ANOVA with Dunnett’s post-hoc test. * P < 0.05, **P < 0.01; **** P < 0.0001. n = 6 independent repeats for all experiments.
References
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