Mechanisms of Antiproliferative Effects of Nobiletin, Scoparone, and Tangeretin Isolated from Citrus reticulata Peel Dichloromethane Extract in Acute Myeloid Leukemia Cells

Abstract

Citrus reticulata Blanco peel is a dominant industrial waste that was recently revalued as a source of bioactive molecules. This study explored its phytochemical and antileukemic potentials. The bioassay-guided fractionation of the dichloromethane extract yielded nobiletin, scoparone, and tangeretin, which were identified spectroscopically. The extract, fractions, and compounds showed antiproliferative effects in both THP-1 and U937 cells, which were employed as in vitro models of acute myeloid leukemia (AML). According to cytofluorimetric analysis, the extract and fractions inhibited cell growth by both apoptosis and necrosis, whereas the single molecules induced apoptosis. This mechanism was mediated by the modulation of BAX and BCL-2 genes in both AML cell lines. Notably, each treatment drove THP-1 and U937 cells into the sub-G0 phase, together with an increase in the cell population in the G1 phase of the cell cycle, both of which were detected cytofluorimetrically. In line with these findings, the extract, fractions, and single molecules counteracted the overexpression of CYCLIN A1 in THP-1 cells while reducing the expression of CYCLIN D2 in U937 cells. Moreover, cell treatments attenuated the invasiveness of AML cells through the upregulation of TIMP-2 at the transcriptional level. Therefore, this study supports pharmaceutical interest in citrus waste for cancer management, providing evidence on its antileukemic potential in vitro.

Keywords: Citrus reticulata; acute myeloid leukemia; apoptosis; cancer; citrus waste; mandarin; nobiletin; peel; scoparone; tangeretin.

Figure 1

Developed TLC plates of crude extracts from C. reticulata peel and ¹H NMR spectra of its n-hexane and dichloromethane extracts. n-Hexane, DCM, MeOH) extracts, and quercetin (QUE) were spotted on TLC plates and visualized under short-wavelength (A) and long-wavelength (B) UV light. The presence of violet, brown, and grey spots was observed in the developed TLC plate sprayed with anisaldehyde reagent (C). The presence of yellow spots was observed in another developed TLC plate after spraying with DPPH reagent (D). The ¹H NMR spectra of n-hexane extract (E) and DCM extract (F) were recorded at 600 MHz using a Bruker AMX Ultrashield NMR spectrometer, with deuterium locking. Chemical shifts are reported in δ (ppm) with CDCl₃ as the internal standard and coupling constants (J) are in Hz.

Figure 2

Antiproliferative activity of dichloromethane and n-hexane extracts from C. reticulata peel in acute myeloid leukemia cells. THP-1 (on the left) and U937 cell lines (on the right) were exposed to different concentrations of DCM (A,B) and n-hexane extracts (C,D) for 24, 48, and 72 h. Cell proliferation was assessed using the MTT assay. Results are expressed as absorbance percentage ± standard error of the mean (SEM). Values detected in treated cells are compared to those in control cells. Each experiment was carried out with eight replicates and repeated three times (n = 24). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. CTRL.

Figure 3

Developed TLC plate of ten fractions obtained from dichloromethane extract. DCM extract and its ten fractions (F1–F10) were spotted on a TLC plate and visualized under short-wavelength (A) and long-wavelength (B) UV light. The presence of brown, yellow, green, and violet spots was detected after spraying the TLC plate with anisaldehyde reagent (C) and then observed under UV light (D).

Figure 4

Antiproliferative activity of combined dichloromethane fractions (F_DCM) in acute myeloid leukemia cell lines. THP-1 (A) and U937 (B) cell lines were exposed to increasing concentrations of F_DCM for 24, 48, and 72 h. Cell proliferation was evaluated using the MTT assay. Results are expressed as absorbance percentage ± standard error of the mean (SEM). Values detected in treated cells are compared to those in control cells. Each experiment was carried out with eight replicates and repeated three times (n = 24). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. CTRL.

Figure 5

Chromatographic separation of combined F4 and F5 dichloromethane fractions (F_DCM) via preparative TLC. Thin bands of F_DCM were applied to a silica gel plate. The developed TLC plate was visualized under short-wavelength (A) and long-wavelength (B) UV light. Three bands (A, B, and C bands in light blue) were separated, and other potential mixtures of compounds were retained in D, E, and F spaces between bands.

Figure 6

Developed TLC plate of A–F samples separated from combined dichloromethane fractions (F_DCM). Samples A–F were spotted on a TLC plate, which was visualized under UV light at a short wavelength (A) and at a long wavelength (B). The purity of samples was further evaluated on a TLC plate after spraying with anisaldehyde reagent and visualization at 365 nm (C).

Figure 7

Compounds identified in combined dichloromethane fractions (F_DCM). The chemical structures of the polymethoxyflavone nobiletin (A); 6,7-dimethoxycoumarin, namely scoparone (B); and 3′-demethoxy nobiletin, known as tangeretin (C) are shown.

Figure 8

Antiproliferative activity of nobiletin, tangeretin, and scoparone in THP-1 and U937 leukemia cells. THP-1 (on the left) and U937 (on the right) cell lines were exposed to increasing concentrations of nobiletin (A,B), tangeretin (C,D), and scoparone (E,F) for 24, 48, and 72 h. Cell proliferation was evaluated using the MTT assay. Results are expressed as absorbance percentage ± standard error of the mean (SEM). Values detected in treated cells are compared to those in untreated cells. Each experiment was carried out in eight replicates and repeated three times (n = 24). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. CTRL.

Figure 9

Cell death mechanisms induced by dichloromethane (DCM) extract and fraction (F_DCM) in acute myeloid leukemia cells. Leukemia THP-1 (A) and U937 cells (B) were exposed to increasing concentrations of DCM extract or F_DCM for 24, 48, and 72 h, or to doxorubicin. Assessment of apoptosis was performed using the Annexin V-FITC/propidium iodide staining assay. On the left, representative density plots (Annexin V vs. PI) are shown. Each plot is subdivided into four quadrants: containing viable cells in Q3, cells in early apoptosis in Q4, cells in late apoptosis in Q2, and cells in necrosis in Q1. Histograms shown on the right express the percentage of cells for each quadrant (Q3: light blue bars; Q4: green bars; Q2: blue bars; Q1: red bars) ± SEM of three experiments performed in triplicate (n = 9).

Figure 10

Cytofluorimetric evaluation of apoptosis in acute myeloid leukemia cells exposed to nobiletin, tangeretin, and scoparone. The assessment of apoptosis was performed using the Annexin V/propidium iodide assay. Representative Annexin V vs. PI dot plots, along with related quantitative graphs, are shown. THP-1 cells (upper) and U937 cells (lower) were treated with different concentrations of nobiletin (NOB, (A,B)), tangeretin (TAN, (C,D)), or scoparone (SCO, (E,F)) for 24, 48, and 72 h. Each plot is divided into four quadrants, containing viable cells in Q4, cells in early apoptosis in Q3, cells in late apoptosis in Q2, and cells in necrosis in Q1. Histograms next to plots report the percentages of cells for each quadrant ± SEM of three independent experiments performed in triplicate (n = 9). The negative control (CTRL) for THP-1 cells treated with NOB and TAN is the same, so CTRL plots shown in (A,C) are the same. Similarly, experiments on U937 cells were performed using the same negative control, so CTRL plots in (B,D,F) are identical.

Figure 11

Expression of apoptosis-related genes in acute myeloid leukemia cells treated with dichloromethane (DCM) extract, combined dichloromethane fractions (F_DCM), nobiletin (NOB), tangeretin (TAN), and scoparone (SCO). Leukemia THP-1 cells (A,C) and U937 cells (B,D) were treated with DCM extract, F_DCM, NOB, TAN, and SCO at the indicated concentrations for 24 h before being processed for gene expression analysis. The mRNA levels of BAX (light red bars) and BCL-2 (light green bars) were quantified via RT-PCR using the 2^−ΔΔCT method and β-actin as an endogenous control. Results are indicated as n-fold change relative to untreated cells (CTRL). Data are expressed as the mean ± SEM of three experiments performed with three replicates (n = 9). * p <0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. CTRL.

Figure 12

Influence on cell cycle of acute myeloid leukemia cells exposed to dichloromethane (DCM) extract and fractions (F_DCM). THP-1 (A) and U937 cells (B) were treated with increasing concentrations of DCM extract or F_DCM for 24, 48, and 72 h. Doxorubicin was used as a positive control. The distribution of cells through cell cycle phases sub-G0 (white bar), G0/G1 (green bar), S (dark yellow bar), and G2/M (cyan bar) was evaluated using the propidium iodide staining assay. On the left, representative plots are shown. On the right, histograms report the percentage of cells for each phase of the cell cycle, expressed as the mean ± SEM of three independent experiments performed in triplicate (n = 9). Vertical lines were set by NovoExpress 1.6.2. software to define the different phases (green line for G0/G1 and light blue for G2/M) based on DNA content. The pink shadow shows the real signal. It matches the black shadow, which shows the signal set by instrument.

Figure 13

Cell cycle analysis in acute myeloid leukemia cells treated with nobiletin (NOB), tangeretin (TAN), or scoparone (SCO). Progression of THP-1 (upper side) and U937 cells (lower side) through the cell cycle phases was assessed after cell exposure to NOB (A,B), TAN (C,D), and SCO (E,F) for 24, 48, and 72 h. Representative plots of three independent experimental sessions are shown on the left of each sub-figure, while histograms, on the right side, report the percentage of cells in sub-G0 (white bar), G0/G1 (green bar), S (dark yellow bar), and G2/M (cyan bar) phases. Data are expressed as the mean ± SEM of three sets of experiments performed in triplicate (n = 9). The negative control (CTRL) for THP-1 cells treated with NOB and TAN is the same, so CTRL plots shown in A and C are the same. Similarly, experiments on U937 cells were performed using same negative control, so CTRL plots in B, D, and F are identical. Vertical lines were set by NovoExpress 1.6.2. software to define the different phases (green line for G0/G1 and light blue for G2/M) based on DNA content. The pink shadow shows the real signal. It matches the black shadow, which shows the signal set by instrument.

Figure 14

Gene expression of CYCLIN A1 and CYCLIN D2 in acute myeloid leukemia cells treated with dichloromethane (DCM) extract, fractions (F_DCM), nobiletin (NOB), tangeretin (TAN), and scoparone (SCO). THP-1 cells or U937 cells were exposed to different concentrations of DCM extract, F_DCM, NOB, TAN, and SCO for 24 h. The mRNA levels of CYCLIN D2 (A) and CYCLIN A1 (B,C) were quantified via RT-PCR using the 2^−ΔΔCT method and β-actin as the housekeeping gene. Results are expressed as n-fold change relative to untreated cells (control, CTRL). Data are represented as the mean ± SEM of three different sets of experiments performed in triplicate (n = 9). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. CTRL.

Figure 15

Dichloromethane (DCM)extract, combined fractions (F_DCM), nobiletin (NOB), tangeretin (TAN), and scoparone (SCO) reduced the invasiveness of THP-1 and U937 cells by upregulating TIMP-2 expression. THP-1 cells (on the left) and U937 cells (on the right) were exposed to different concentrations of DCM extract, F_DCM, NOB, TAN, and SCO for 24 h. For the invasion assay (A,B), leukemia cells migrating through the filter of Matrigel invasion chambers were counted and the invasion rate is expressed as a percentage relative to the control. For RT-PCR analysis (C,D), the mRNA levels of TIMP-2 were quantified using the 2^−ΔΔCT method and β-actin as the housekeeping gene. Results of qPCR are expressed as n-fold change relative to untreated cells (control, CTRL). All data are represented as the mean ± SEM of three different sets of experiments performed in triplicate (n = 9). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. CTRL.