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Study of Malformin C, a Fungal Source Cyclic Pentapeptide, as an Anti-Cancer Drug.

Wang J, Jiang Z, Lam W, Gullen EA, Yu Z, Wei Y, Wang L, Zeiss C, Beck A, Cheng EC, Wu C, Cheng YC, Zhang Y - PLoS ONE (2015)

Bottom Line: This inhibition was explicated by Malformin C's effect on G2/M arrest.Overall, we conclude that Malformin C arrests Colon 38 cells in G2/M phase and induces multiple forms of cell death through necrosis, apoptosis and autophagy.Malformin C has potent cell growth inhibition activity, but the therapeutic index is too low to be an anti-cancer drug.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, United States of America.

ABSTRACT
Malformin C, a fungal cyclic pentapeptide, has been claimed to have anti-cancer potential, but no in vivo study was available to substantiate this property. Therefore, we conducted in vitro and in vivo experiments to investigate its anti-cancer effects and toxicity. Our studies showed Malformin C inhibited Colon 38 and HCT 116 cell growth dose-dependently with an IC50 of 0.27±0.07μM and 0.18±0.023μM respectively. This inhibition was explicated by Malformin C's effect on G2/M arrest. Moreover, we observed up-regulated expression of phospho-histone H2A.X, p53, cleaved CASPASE 3 and LC3 after Malformin C treatment, while the apoptosis assay indicated an increased population of necrotic and late apoptotic cells. In vivo, the pathological study exhibited the acute toxicity of Malformin C at lethal dosage in BDF1 mice might be caused by an acute yet subtle inflammatory response, consistent with elevated IL-6 in the plasma cytokine assay. Further anti-tumor and toxicity experiments proved that 0.3mg/kg injected weekly was the best therapeutic dosage of Malformin C in Colon 38 xenografted BDF1 mice, whereas 0.1mg/kg every other day showed no effect with higher resistance, and 0.9mg/kg per week either led to fatal toxicity in seven-week old mice or displayed no advantage over 0.3mg/kg group in nine-week old mice. Overall, we conclude that Malformin C arrests Colon 38 cells in G2/M phase and induces multiple forms of cell death through necrosis, apoptosis and autophagy. Malformin C has potent cell growth inhibition activity, but the therapeutic index is too low to be an anti-cancer drug.

No MeSH data available.


Related in: MedlinePlus

Expression of CASPASE 3 and LC3 and apoptosis assay in cells treated with Malformin C.(A, B) The expression of cleaved CASPASE 3, total CASPASE 3, LC3AI and LC3AII in Colon 38 and HCT 116 cells treated with different concentrations of Malformin C (0μM, 0.14μM, 0.27μM, 0.54μM for 24 hours was tested by Western blot, with β-Actin expression as an internal control. During autophagy process, LC3AI will be converted into LC3AII to be functional, so we analyzed LC3AII expression for autophagy process. (C) Malformin C led to late apoptosis and necrosis after 24-hour treatment examined by apoptosis assay. Untreated or Malformin C-treated Colon 38 cells (2×106) were stained with Alexa Fluor 488 annexin V and PI and processed by flow cytometer. The percentage of early apoptotic population (lower right panel), late apoptotic population (upper right panel) and necrotic population (upper left panels) is shown in the graph. CPT, a known apoptosis-inducing agent, was used as a control. Three independent experiments were performed with duplicates for each condition, and one typical result is shown here.
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pone.0140069.g004: Expression of CASPASE 3 and LC3 and apoptosis assay in cells treated with Malformin C.(A, B) The expression of cleaved CASPASE 3, total CASPASE 3, LC3AI and LC3AII in Colon 38 and HCT 116 cells treated with different concentrations of Malformin C (0μM, 0.14μM, 0.27μM, 0.54μM for 24 hours was tested by Western blot, with β-Actin expression as an internal control. During autophagy process, LC3AI will be converted into LC3AII to be functional, so we analyzed LC3AII expression for autophagy process. (C) Malformin C led to late apoptosis and necrosis after 24-hour treatment examined by apoptosis assay. Untreated or Malformin C-treated Colon 38 cells (2×106) were stained with Alexa Fluor 488 annexin V and PI and processed by flow cytometer. The percentage of early apoptotic population (lower right panel), late apoptotic population (upper right panel) and necrotic population (upper left panels) is shown in the graph. CPT, a known apoptosis-inducing agent, was used as a control. Three independent experiments were performed with duplicates for each condition, and one typical result is shown here.

Mentions: The expression of CASPASE 3 and LC3 was also examined by Western blot. During autophagy, LC3AI is converted to LC3AII through lipidation, and LC3AII serves as an indicator of autophagy. So we analyzed LC3AII expression to test Malformin C’s effect on autophagy. After 24-hour treatment of Malformin C, we observed a dose-dependent up-regulation of cleaved CASPASE 3 in HCT 116 cells, and a dose-dependent up-regulation of LC3AII in both Colon 38 and HCT 116 cells (Fig 4A and 4B). But there was no dose-response for the increased expression of cleaved CASPASE 3 in Colon 38 cells, and it could due to the fact that some apoptotic cells detached from the plate during culture and were lost when the media was removed. No significant changes of cleaved CASPASE 3 and LC3AII expression were detected after 4-hour and 8-hour treatment (S5 Fig). To further investigate the death process of those DNA-damaged cells, we conducted the apoptosis assay and found a significantly larger group of late apoptotic cells and necrotic cells after Malformin C exposure, while the percentage of early apoptotic cells showed no difference from the control (Fig 3E). All in all, we found that 24-hour treatment of Malformin C could cause DNA damage and lead to cell death through apoptosis, autophapy and necrosis.


Study of Malformin C, a Fungal Source Cyclic Pentapeptide, as an Anti-Cancer Drug.

Wang J, Jiang Z, Lam W, Gullen EA, Yu Z, Wei Y, Wang L, Zeiss C, Beck A, Cheng EC, Wu C, Cheng YC, Zhang Y - PLoS ONE (2015)

Expression of CASPASE 3 and LC3 and apoptosis assay in cells treated with Malformin C.(A, B) The expression of cleaved CASPASE 3, total CASPASE 3, LC3AI and LC3AII in Colon 38 and HCT 116 cells treated with different concentrations of Malformin C (0μM, 0.14μM, 0.27μM, 0.54μM for 24 hours was tested by Western blot, with β-Actin expression as an internal control. During autophagy process, LC3AI will be converted into LC3AII to be functional, so we analyzed LC3AII expression for autophagy process. (C) Malformin C led to late apoptosis and necrosis after 24-hour treatment examined by apoptosis assay. Untreated or Malformin C-treated Colon 38 cells (2×106) were stained with Alexa Fluor 488 annexin V and PI and processed by flow cytometer. The percentage of early apoptotic population (lower right panel), late apoptotic population (upper right panel) and necrotic population (upper left panels) is shown in the graph. CPT, a known apoptosis-inducing agent, was used as a control. Three independent experiments were performed with duplicates for each condition, and one typical result is shown here.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4635020&req=5

pone.0140069.g004: Expression of CASPASE 3 and LC3 and apoptosis assay in cells treated with Malformin C.(A, B) The expression of cleaved CASPASE 3, total CASPASE 3, LC3AI and LC3AII in Colon 38 and HCT 116 cells treated with different concentrations of Malformin C (0μM, 0.14μM, 0.27μM, 0.54μM for 24 hours was tested by Western blot, with β-Actin expression as an internal control. During autophagy process, LC3AI will be converted into LC3AII to be functional, so we analyzed LC3AII expression for autophagy process. (C) Malformin C led to late apoptosis and necrosis after 24-hour treatment examined by apoptosis assay. Untreated or Malformin C-treated Colon 38 cells (2×106) were stained with Alexa Fluor 488 annexin V and PI and processed by flow cytometer. The percentage of early apoptotic population (lower right panel), late apoptotic population (upper right panel) and necrotic population (upper left panels) is shown in the graph. CPT, a known apoptosis-inducing agent, was used as a control. Three independent experiments were performed with duplicates for each condition, and one typical result is shown here.
Mentions: The expression of CASPASE 3 and LC3 was also examined by Western blot. During autophagy, LC3AI is converted to LC3AII through lipidation, and LC3AII serves as an indicator of autophagy. So we analyzed LC3AII expression to test Malformin C’s effect on autophagy. After 24-hour treatment of Malformin C, we observed a dose-dependent up-regulation of cleaved CASPASE 3 in HCT 116 cells, and a dose-dependent up-regulation of LC3AII in both Colon 38 and HCT 116 cells (Fig 4A and 4B). But there was no dose-response for the increased expression of cleaved CASPASE 3 in Colon 38 cells, and it could due to the fact that some apoptotic cells detached from the plate during culture and were lost when the media was removed. No significant changes of cleaved CASPASE 3 and LC3AII expression were detected after 4-hour and 8-hour treatment (S5 Fig). To further investigate the death process of those DNA-damaged cells, we conducted the apoptosis assay and found a significantly larger group of late apoptotic cells and necrotic cells after Malformin C exposure, while the percentage of early apoptotic cells showed no difference from the control (Fig 3E). All in all, we found that 24-hour treatment of Malformin C could cause DNA damage and lead to cell death through apoptosis, autophapy and necrosis.

Bottom Line: This inhibition was explicated by Malformin C's effect on G2/M arrest.Overall, we conclude that Malformin C arrests Colon 38 cells in G2/M phase and induces multiple forms of cell death through necrosis, apoptosis and autophagy.Malformin C has potent cell growth inhibition activity, but the therapeutic index is too low to be an anti-cancer drug.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, United States of America.

ABSTRACT
Malformin C, a fungal cyclic pentapeptide, has been claimed to have anti-cancer potential, but no in vivo study was available to substantiate this property. Therefore, we conducted in vitro and in vivo experiments to investigate its anti-cancer effects and toxicity. Our studies showed Malformin C inhibited Colon 38 and HCT 116 cell growth dose-dependently with an IC50 of 0.27±0.07μM and 0.18±0.023μM respectively. This inhibition was explicated by Malformin C's effect on G2/M arrest. Moreover, we observed up-regulated expression of phospho-histone H2A.X, p53, cleaved CASPASE 3 and LC3 after Malformin C treatment, while the apoptosis assay indicated an increased population of necrotic and late apoptotic cells. In vivo, the pathological study exhibited the acute toxicity of Malformin C at lethal dosage in BDF1 mice might be caused by an acute yet subtle inflammatory response, consistent with elevated IL-6 in the plasma cytokine assay. Further anti-tumor and toxicity experiments proved that 0.3mg/kg injected weekly was the best therapeutic dosage of Malformin C in Colon 38 xenografted BDF1 mice, whereas 0.1mg/kg every other day showed no effect with higher resistance, and 0.9mg/kg per week either led to fatal toxicity in seven-week old mice or displayed no advantage over 0.3mg/kg group in nine-week old mice. Overall, we conclude that Malformin C arrests Colon 38 cells in G2/M phase and induces multiple forms of cell death through necrosis, apoptosis and autophagy. Malformin C has potent cell growth inhibition activity, but the therapeutic index is too low to be an anti-cancer drug.

No MeSH data available.


Related in: MedlinePlus