Anticancer Potential of Damnacanthal and Nordamnacanthal from Morinda elliptica Roots on T-lymphoblastic Leukemia Cells

Abstract

Background: This study reports on the cytotoxic properties of nordamnacanthal and damnacanthal, isolated from roots of Morinda elliptica on T-lymphoblastic leukaemia (CEM-SS) cell lines.

Methods: MTT assay, DNA fragmentation, ELISA and cell cycle analysis were carried out.

Results: Nordamnacanthal and damnacanthal at IC₅₀ values of 1.7 μg/mL and10 μg/mL, respectively. At the molecular level, these compounds caused internucleosomal DNA cleavage producing multiple 180-200 bp fragments that are visible as a "ladder" on the agarose gel. This was due to the activation of the Mg²⁺/Ca²⁺-dependent endonuclease. The induction of apoptosis by nordamnacanthal was different from the one induced by damnacanthal, in a way that it occurs independently of ongoing transcription process. Nevertheless, in both cases, the process of dephosphorylation of protein phosphates 1 and 2A, the ongoing protein synthesis and the elevations of the cytosolic Ca²⁺ concentration were not needed for apoptosis to take place. Nordamnacanthal was found to have a cytotoxic effect by inducing apoptosis, while damnacanthal caused arrest at the G0/G1 phase of the cell cycle.

Conclusion: Damnacanthal and nordamnacanthal have anticancer properties, and could act as potential treatment for T-lymphoblastic leukemia.

Keywords: CEM-SS; G0/G1 arrest; Mg2+/Ca2+-dependent endonuclease; anticancer; apoptosis; cytotoxic; damnacanthal; nordamnacanthal.

Figure 1

Chemical structure of nordamnacanthal (A) and damnacanthal (B).

Figure 2

The percentage of viability (relative to control) of T-lymphoblastic leukaemia (CEM-SS) cells treated with different concentrations of nordamnacanthal (A) and damnacanthal (B) for a 72 h period, determined using the MTT assay. Control cultures were not treated with nordamnacanthal or damnacanthal. Each data point represents the mean of three independent experiments, and vertical lines through the data points indicate standard deviation.

Figure 3

Effects of different concentrations of nordamnacanthal at different hours on DNA fragmentation in CEM-SS cells. Lane M: Marker (HindIII digest of lambda DNA).

Figure 4

The involvement of Ca²⁺/Mg²⁺-dependent endonuclease and protein synthesis in nordamnacanthal-induced apoptosis in CEM-SS cells at 24 h. Zinc sulphate and cycloheximide failed to prevent apoptosis induced by 10 µg/mL of nordamnacanthal at 24 h. Lane a: control. Lane b: nordamnacanthal (10 µg/mL). Lane c: zinc sulphate (0.1 mM). Lane d: nordamnacanthal (10 µg/mL) + zinc sulphate (0.1 mM). Lane e: cycloheximide (10 µg/mL). Lane f: nordamnacanthal (10 µg/mL) + cycloheximide (10 µg/mL). Lane M: Marker (HindIII digest of lambda DNA).

Figure 5

The involvement of RNA synthesis in nordamnacanthal-induced apoptosis in CEM-SS cells at 6 and 8 h. Actinomycin D failed to prevent apoptosis induced by 30 µg/mL of nordamnacanthal at 6 and 8 h. Lane a: control. Lane b: nordamnacanthal (30 µg/mL) (6 h-treatment). Lane c: actinomycin D (1 µg/mL) (6 h-treatment). Lane d: nordamnacanthal (30 µg/mL) + actinomycin D (1 µg/mL) (6 h-treatment). Lane e: nordamnacanthal (30 µg/mL) (8 h treatment). Lane f: actinomycin D (1 µg/mL) (8 h-treatment). Lane g: nordamnacanthal (30 µg/mL) + actinomycin D (1µg/mL) (8 h-treatment). Lane M: Marker (HindIII digest of lambda DNA).

Figure 6

The involvement of phosphatases and RNA synthesis in nordamnacanthal-induced apoptosis in CEM-SS cells at 24 h. Okadaic acid and actinomycin D failed to prevent apoptosis induced by 10 µg/mL of nordamnacanthal at 24 h. Lane a: control. Lane b: nordamnacanthal (10 µg/mL). Lane c: okadaic acid (100 nM). Lane d: nordamnacanthal (10 µg/mL) + okadaic acid (100 nM). Lane e: actinomycin D (1 µg/mL). Lane f: nordamnacanthal (10 µg/mL) + actinomycin D (1 µg/mL). Lane M: Marker (HindIII digest of lambda DNA).

Figure 7

The involvement of an increase in cytosolic calcium concentration in nordamnacanthal-induced apoptosis in CEM-SS cells at 24 h. EGTA failed to prevent apoptosis induced by 10 µg/mL of nordamnacanthal at 24 h. Lane a: control. Lane b: nordamnacanthal (10 µg/mL). Lane c: EGTA (1 mM). Lane d: nordamnacanthal (10 µg/mL) + EGTA (1 mM). Lane e: EGTA (8 mM). Lane f: nordamnacanthal (10 µg/mL) + EGTA (8 mM). Lane M: Marker (HindIII digest of lambda DNA).

Figure 8

Effects of different concentrations of damnacanthal at different hours on DNA fragmentation in CEM-SS cells. Lane M: Marker (HindIII digest of lambda DNA).

Figure 9

The involvement of Ca²⁺/Mg²⁺-dependent endonuclease and protein synthesis in damnacanthal-induced apoptosis in CEM-SS cells at 24 h. Zinc sulphate and cycloheximide prevented apoptosis induced by 30 µg/mL of damnacanthal at 24 h. Lane a: control. Lane b: damnacanthal (30 µg/mL). Lane c: zinc sulphate (0.1 mM). Lane d: damnacanthal (30 µg/mL) + zinc sulphate (0.1 mM). Lane e: cycloheximide (10 µg/mL). Lane f: damnacanthal (30 µg/mL) + cycloheximide (10 µg/mL). Lane M: Marker (HindIII digest of lambda DNA).

Figure 10

The involvement of RNA synthesis in damnacanthal-induced apoptosis in CEM-SS cells at 24 h. Actinomycin D failed to prevent apoptosis induced by 30 µg/mL of damnacanthal at 24 h. Lane a: control. Lane b: damnacanthal (30 µg/mL). Lane c: actinomycin D (1 µg/mL). Lane d: damnacanthal (30 µg/mL) + actinomycin D (1 µg/mL). Lane M: Marker (HindIII digest of lambda DNA).

Figure 11

The involvement of RNA synthesis in damnacanthal-induced apoptosis in CEM-SS cells at 24 h. Actinomycin D failed to prevent apoptosis induced by 30 µg/mL of damnacanthal at 24 h. Lane a: control. Lane b: damnacanthal (30 µg/mL). Lane c: actinomycin D (10 µg/mL). Lane d: damnacanthal (30 µg/mL) + actinomycin D (10 µg/mL). Lane M: Marker (HindIII digest of lambda DNA).

Figure 12

The involvement of increase in cytosolic calcium concentration and phosphatases in damnacanthal-induced apoptosis in CEM-SS cells at 24 h. EGTA and okadaic acid failed to prevent apoptosis induced by 30 µg/mL of damnacanthal at 24 h. Lane a: control. Lane b: damnacanthal (30 µg/mL). Lane c: EGTA (0.5 mM). Lane d: damnacanthal (30 µg/mL) + EGTA (0.5 mM). Lane e: okadaic acid (100 nM). Lane f: damnacanthal (30 µg/mL) + okadaic acid (100 nM). Lane M: Marker (HindIII digest of lambda DNA).

Figure 13

The involvement of increase in cytosolic calcium concentration in damnacanthal-induced apoptosis in CEM-SS cells at 24 h. EGTA failed to prevent apoptosis induced by 30 µg/mL of damnacanthal at 24 h. Lane a: control. Lane b: damnacanthal (30 µg/mL). Lane c: EGTA (1mM). Lane d: damnacanthal (30 µg/mL) + EGTA (1mM). Lane M: Marker (HindIII digest of lambda DNA).

Figure 14

Detection of nucleosomes in cytoplasmic fractions of cell lysates at different hours of experiments. CEM-SS cells were treated with 30 μg/mL of indicated compounds. Control cultures were not treated with nordamnacanthal or damnacanthal. 20 μL of cell lysates were analyzed in the ELISA.

Figure 15

Enrichment of nucleosomes in cytoplasmic fractions of cell lysates at different hours of experiments. CEM-SS cells were treated with 30 μg/mL of indicated compounds. Control cultures were not treated with nordamnacanthal or damnacanthal. 20 μL of cell lysates were analyzed in the ELISA.

Figure 16

Cell cycle distribution of CEM-SS cells after 24 h and 48 h incubation with (A) nordamnacanthal and (B) damnacanthal at their respective IC₅₀ values.