Fig 1.
Cell cycle analysis of Colon 38 cells treated by Malformin C and its combinations.
(A) Chemical structure of Malformin C. Malformin C is a member of Malformins, a group of fungal cyclic pentapeptides. Its chemical formula is C23H39O5N5S2 with a molecular weight of 529.7. (B) Cell cycle progression of Colon 38 cells exposed to an increasing concentration of Malformin C for 24 hours. (C) Cell cycle progression of Colon 38 cells exposed to an increasing concentration of Malformin C for 48 hours. (D) The dose-dependent accumulation of G2-M phase Colon 38 cells treated by Malformin C at concentrations of 90 nM, 270 nM and 810 nM. Compared to the control group, the symbol Δ represented P<0.05, while * represented P<0.01. (E) Cell cycle analysis of Colon 38 cells treated with combinations of Malformin C and SN38. All the cells were exposed to respective compounds indicated above the graph for 24 hours and the analysis was done in duplicate. When treated with SN38, no significant changes of cell cycle progression were observed without or with different dosage of Malformin C.
Table 1.
Characteristics of different KB resistant cell lines.
Table 2.
Growth inhibition of KB and its drug-resistant cells by Malformin C and anti-cancer drugs.
Table 3.
Multidrug resistance of Malformin C and reversal effects of Verapamil.
Fig 2.
Immunofluorescence micrographs of the p53, cleaved CASPASE 3 and LC3 in Malformin C treated Colon 38 cells.
The expression level (green fluorescence) was probed with specific monoclonal p53, cleaved CASPASE 3 and LC3 antibodies respectively, followed by FITC-conjugated anti-mouse IgG. Cytoplasmic Actin (red fluorescence) was counterstained with rhodamine phalloidin. Immunofluorescence micrographs are taken by confocal microscope. (A) Experiment design-Malformin C was given for 24h and stained at 24h, for 24h and stained at 48h, for 48h and stained at 48h, respectively. (B) Immunofluorescence staining at 24h after 24h treatment. (C) Immunofluorescence staining at 48h after 24h treatment. (D) Immunofluorescence staining at 48h after 48h treatment. (E) Expression of p53, cleaved CASPASE 3 and LC3 after three ways of administration of different concentration of Malformin C based on confocal microscopy results.
Fig 3.
Expression of phosphorylated and total H2A.X in Colon 38 and HCT 116 cells treated with Malformin C.
(A, C) The expression of phosphorylated and total H2A.X in Colon 38 cells treated with different concentrations of Malformin C (0μM, 0.14μM, 0.27μM, 0.54μM) and Hydroxyurea (1mM, 2mM) for 2-hour, 4 hours, 8 hours and 24 hours tested by Western blot, with β-Actin expression as an internal control. Hydroxyurea was used as positive control. H2A.X is phosphorylated at Ser139. (B, D) The expression of phosphorylated and total H2A.X in HCT116 cells treated with different concentrations of Malformin C (0μM, 0.14μM, 0.27μM, 0.54μM) and Hydroxyurea (1mM, 2mM) for 2 hours, 4 hours, 8 hours and 24 hours tested by Western blot, with β-Actin expression as an internal control. Hydroxyurea was used as positive control. H2A.X is phosphorylated at Ser139.
Fig 4.
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.
Fig 5.
Pathological investigation of Malformin C treated mice.
(A) Observation of mice prior to death injected with 1.8mg/kg Malformin C. The mice exhibited hunched posture, huddling and piloerection. (B) Control mice injected with PBS were active, with normal haircoats. (C) The parameter indicating the most different between treated and control mice was AST (aspartate aminotransferase). AST elevation may be lower than reported due to a hemolysis 3+, but is still abnormal. (D) There were also mild differences in ALT (alanine amino transferase) values between treated and control mice. (E) ALP (alkaline phosphatase) could be elevated with cholestasis, but in this case, the average value for the treated and control mice was identical, and thus not considered to be a significant finding. (F) Mouse IL-6 level in the plasma of Colon 38 bearing BDF-1 mice. The left graph showed a dose dependent increase of mouse IL-6 in the plasma 6 hours after 0.9mg/kg, 0.3mg/kg and 0.1mg/kg Malformin C injection compared to PBS group, while the right graph showed similar effects 4 hours after 0.9mg/kg, 0.3mg/kg Malformin C injection. (G) Mouse TNF level in the plasma of Colon 38 bearing BDF-1 mice. No significant changes were shown either 6 hours or 4 hours before and after different concentrations of Malformin C injection compared to PBS injection group.
Fig 6.
The anti-cancer effects and in vivo toxicity of Malformin C.
(A) Survival of BDF-1 mice treated with Malformin C. All the mice in 2.6mg/kg and 1.8mg/kg group died within 1 day after injection, and all the mice in 0.3mg/kg and 0.1mg/kg survived throughout the experiment. There were 5 mice in each group and the week age indicated was that at the beginning of the experiment. One of the mice in 0.9mg/kg qw 7-week old group died by accident and was therefore excluded from the graph. Malformin C was given by i.p., qw means every week, qod means every other day. (B) Tumor growth of Colon 38 bearing BDF-1 mice treated with Malformin C. All the mice were 7 weeks old on Day 0. Control group was treated with 0.1ml/10g PBS (the intake volume was the same for all groups), 0.9mg/kg group was injected with Malformin C on Day 1, 0.3mg/kg q.w. group was treated with 0.3mg/kg on Day 1 and treated with 0.9mg/kg on Day 9 in order to confirm age and toxicity effect, 0.3mg/kg q.o.d. group was treated with 0.3mg/kg Malformin C on Day 1, 3, 5, 9, 11, 13 and 16. (C) Body weight changes of corresponding groups in A were measured. (D) Tumor growth of Colon 38 bearing BDF-1 mice treated with Malformin C. All the mice were 8 weeks old on Day 0. Control group was treated with 0.1ml/10g PBS, 0.9mg/kg group was injected with Malformin C on Day 1 and 9, 0.3mg/kg q.w. group was treated with 0.3mg/kg on Day 1 and 9, 0.1mg/kg q.o.d. group was treated with 0.1mg/kg Malformin C on Day 1, 3, 5 and 9. (E) Body weight changes of corresponding groups in C were measured.