Effects of Ligusticum porteri (Osha) Root Extract on Human Promyelocytic Leukemia Cells

Abstract

Background:

Ligusticum porteri roots have been traditionally used in folk medicine, but the scientific basis is unclear.

Objective:

To investigate the cytotoxicity, antioxidant, and immunomodulatory effects of L. porteri root extract on human promyelocytic leukemia (HL-60) cells and H₂O₂-induced oxidative damaged HL-60 cells.

Materials and Methods:

HL-60 cells were incubated with different concentrations of root extract, and cells were harvested for viability assays on day 3 and 7. Cytokine levels (interferon-gamma [IFN-γ], interleukin-2 [IL-2], and interleukin-10 [IL-10]) and antioxidant indexes (malondialdehyde [MDA], reduced glutathione [GSH], superoxide dismutase [SOD], and catalase [CAT]) in H₂O₂-induced-stressed HL-60 were measured after 2 days.

Results:

The viability of HL-60 challenged with H₂O₂ declined by 42% compared to unstressed cells. After 7 days of incubation with 200 or 400 μg/mL L. porteri, the viability of HL-60 cells was two-fold higher than the control. Stressed HL-60 cells treated with 100, 200, and 400 μg/mL L. porteri reduced the lipid peroxidation by 12%–13%. We noted an increase in GSH levels, SOD and CAT activities in stressed HL-60 supplemented with 400 μg/mL root extract. Treatment with 400 μg/mL L. porteri significantly (P < 0.05) increased IFN-γ and IL-2 in H₂O₂-challenged cells.

Conclusion:

Our data do not support the use of the extract as an antiproliferation and differentiation therapy for acute promyelocytic leukemia. The protective function of L. porteri root extract against oxidative stress could occur through increasing GSH and higher expression of antioxidant enzymes.

SUMMARY

• Findings from this study may not support the use of Ligusticum porteri root extract as an antiproliferation and differentiation therapy for acute promyelocytic leukemia

• Our data suggest that L. porteri root extract may be a potential antioxidant with protective effect against the oxidation of reduced glutathione (GSH)

• Treatment with L. porteri root extract may be effective in preventing oxidative damage through increasing the activities of antioxidant enzymes (superoxide dismutase [SOD] and catalase [CAT]) in acute promyelocytic leukemia cells.

INTRODUCTION

Hispanics and Native Americans have used Ligusticum porteri preparations for many years for the treatment of many diseases.[1] L. porteri enhances the immune system, stimulates appetite, and improves gastrointestinal discomforts such as indigestion and stomach upset associated with vomiting.[2] L. porteri is used to treat acute influenza, acute bronchial pneumonia with dyspnea, and leukocytosis.[3,4,5]

Phytochemical studies conducted identified butylidenephthalide and ligustilide as the most common constituents isolated from Ligusticum.[6] Butylidenephthalide is effective as an anti-angina, antihypertensive, antioxidative, antiplatelet, antispasmodic, and vasodilator.[6,7] Ligustilide has antimicrobial activities against Gram-positive, Gram-negative, and yeast organisms. The essential oil (100 μg/mL) prepared from the roots increased the antimicrobial activity of antibiotic norfloxacin against the norfloxacin-resistant strain of Staphylococcus aureus.[8] Ligusticum spp. is employed in traditional medicine to boost the immune system.[4,5,6,9] Z-ligustilide was reported to possess anti-inflammatory activity.[6] Doses of 20 mg/kg/day of Z-ligustilide reduced pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), vascular endothelial growth factor-α, and IL-17 in endotoxin-infected mice within 24 h. The suppression of these pro-inflammatory mediators has been found to reduce the severity of the inflammatory reaction.[10] Z-ligustilide and Senkyunolide A, which make up 50% of organic constituents in Ligusticum species, suppressed the production of TNF-α during inflammation.[11,12]

Most inflammatory diseases are treated with conventional steroidal and nonsteroidal anti-inflammatory drugs.[13] The use of medicinal plant preparations in the management of diseases is on the increase. However, the scientific basis for the traditional use of many plant preparations in the treatment of many diseases including cancer is not clear. For example, it was reported that L. porteri extract does not exert anticancer activity on breast and colon cancer cells at a concentration of 50 μg/mL.[14] The HL-60 cells were established in 1977 from the peripheral blood of a patient with acute myeloid leukemia.[15] It has been used as a model in many studies of inflammatory cells because this cell line can be induced to differentiate into granulocytes in vitro.[16] We have previously reported the cytotoxicity, antioxidative and immune-modulatory effects of L. porteri root extract on human peripheral blood lymphocytes.[17] In this study, we used similar protocols to evaluate the effects of L. porteri root extract at a concentration range of 50–400 μg/mL on oxidative stress and inflammation in human promyelocytic leukemia (HL-60) cells and the probable mechanism of action.

MATERIALS AND METHODS

Preparation of Ligusticum porteri root extract

The extract was prepared by the following Beltran's method with modifications.[18] About 30 g of dry L. porteri root was pulverized and mixed with 300 mL of 40% ethanol. The mixture was sieved through a cheese-cloth, followed by a final filtration with 0.20 μm membrane. The filtrate was dried under vacuum and stored at −20°C for further use. The dried sample was weighed and then dissolved in 0.1% dimethyl sulfoxide (DMSO) (Corning Cellgro, Virginia, USA) at a concentration of 440 μg/mL (stock solution). Final working concentrations of the root extract at 50, 100, 200, 400, and 0 μg/mL (the control containing only DMSO) were prepared right before the experiments.[17,19]

Preparation of cultured human promyelocytic leukemia cells

HL-60 cells (ATCC, Virginia, USA) at the seeding concentration of 10⁵ cells/mL were suspended in Iscove's Modified Dulbecco's Medium (ATCC, Virginia, USA) supplemented with 20% (v/v) fetal bovine serum (ATCC, Virginia, USA). The HL-60 cells were incubated at 37°C in humidified atmosphere containing 5% CO₂. When the cell concentration reached 10⁶ cells/mL, the cell culture was diluted for subculturing according to the manufacturer's instruction (ATCC, Virginia, USA). Hydrogen peroxide (50 μM)[20] was used to induce oxidative stress in HL-60 cultures and cell concentration was determined by 0.4% Trypan blue as previously described.[17]

Cytotoxicity assays

One hundred microliters of cell suspension (10⁶ cells/mL) was seeded in each well in a 96-well plate and preincubated for 24 h in a humidified incubator at 37°C with 5% CO₂. Subsequently, 10 μL of L. porteri was added to test for cytotoxicity (ratio of root extract and cell suspension is 1:10 v/v so that the final concentrations of L. porteri were 50, 100, 200, and 400 μg/mL, respectively). The vehicle (0.1% DMSO) was used as the control. The plate was incubated for 3 and 7 days. Cytotoxicity was determined using CCK-8 solution. Ten microliters of CCK-8 solution was added to each well of the plate and incubated for 4 h at 37°C. The absorbance of formazan was measured at 450 nm (Technical manual Cell Counting Kit-8, Maryland, USA) as previously described.[17]

Lipid peroxidation, reduced glutathione levels and antioxidant enzymes

After treatment of HL-60 cells with 50, 100, 200, 400, and 0 μg/mL (control) of L. porteri root extract, the cell pellets were harvested for the assays of lipid peroxidation and GSH levels,[21,22,23,24] and SOD and CAT activities[21,25] as previously described.[17]

Determination of interferon-gamma, interleukin-2, and interleukin-10

Cultures of HL-60 were induced to differentiate by addition of 1.0 μg/mL all–trans retinoic acid (Sigma-Aldrich, Minnesota, USA) and incubated for 4 days.[26] The differentiated HL-60 cells (10⁶ cells/mL) were exposed to 50 μM H₂O₂, followed by treatment with 400 μg/mL L. porteri extract or the control. After 2 days of incubation, the supernatants were removed for analyses of cytokines. Cytokine levels of IFN-γ, IL-2, and IL-10 in the culture supernatants were determined using commercial enzyme-linked immunosorbent assays (ELISA) obtained from Thermo Scientific (Instructions for human IFN-γ, IL-2, and IL-10 ELISA kits, Illinois, USA).

Statistical analysis

Data are presented as mean ± standard error of the mean. Data were obtained from three separate experiments and each experiment included the control and four other concentrations of L. porteri extracts performed in triplicate.[17] The results among different concentrations were evaluated by one-way ANOVA (P < 0.05). Duncan's multiple range test at significance level P < 0.05 was used to test for significant difference among the means.

RESULTS

Figure 1 shows that treatment with L. porteri root extract at doses higher than 100 μg/mL enhanced the viability of HL-60 cells after 7 days of incubation. Growth of HL-60 cells in the control reached a peak on day 3, followed by a decrease in their viability on day 7 of incubation. The viability of HL-60 cells treated with 100 μg/mL of L. porteri extract increased by 31% as compared to the control after 7 days of incubation. The HL-60 cells treated with 200 μg/mL and 400 μg/mL of L. porteri extract showed a 2-fold increase in viability after 7 days of incubation.

Figure 1.

Change in the viability of human promyelocytic leukemia cells cells treated with different concentrations of Ligusticum porteri during 7 days of incubation. Figures that share different letters are significantly different (P < 0.05)

Figure 2 shows that H₂O₂ exerted a cytotoxic effect to HL-60 cells after 7 days of incubation. The viability of HL-60 cells was declined by 42% after 7 days of incubation (P < 0.05).

Figure 2.

The effect of 50 μM H₂O₂ on human promyelocytic leukemia cells after 7 days. Figures that share different letters are significantly different (P < 0.05)

Figure 3 shows that treatment with 400 μg/mL L. porteri root extract significantly ameliorated the adverse effects of H₂O₂ in HL-60 cells. Only treatment with 400 μg/mL L. porteri root extract was effective in boosting the HL-60 cell survival; it increased by 30% after 7 days of incubation compared to the control. Other lower concentrations of the root extract (≤200 μg/mL) did not considerably relieve the deleterious effect of H₂O₂ after a period of incubation. On day 0, viability of HL-60 cells was not detected in the groups treated with different concentrations of L. porteri root extract. However, HL-60 cell viability was seen in treated groups on day 3 and 7.

Figure 3.

Change in the viability of H₂O₂-induced-stress human promyelocytic leukemia cells after treatment with Ligusticum porteri root extract for 7 days. Figures that share different letters are significantly different (P < 0.05)

Table 1 shows that HL-60 cells treated with the extract doses of 100, 200, and 400 μg/mL reduced lipid peroxidation by 12%–13%.

Table 1.

Effects of Ligusticum porteri on lipid peroxidation in H₂O₂-induced-stress human promyelocytic leukemia cells after 2 days

Table 2 shows that treatments with 200 μg/mL and 400 μg/mL of L. porteri root extract elevated GSH levels by approximately 29%–30% while other treatments with lower concentration showed 7%–8% elevations.

Table 2.

Effects of Ligusticum porteri root extract on glutathione content in H₂O₂-induced-stress human promyelocytic leukemia cells after 2 days

Table 3 shows that treatment with L. porteri at the concentration as low as 100 μg/mL enhanced the activities of SOD (P < 0.05). The antioxidant effect of L. porteri on the enzyme activity was greater with increasing concentrations of root extract. The modulatory effect of L. porteri on SOD activity was seen when the root extract was above 100 μg/mL.

Table 3.

Effects of Ligusticum porteri root extract on antioxidant enzyme superoxide dismutase activity in H₂O₂-induced-stress human promyelocytic leukemia cells after 2 days

Table 4 shows that CAT activity in stressed HL-60 cells increased by 2.5-fold after treatment with L. porteri root extract at a dose above 200 μg/mL.

Table 4.

Effects of Ligusticum porteri root extract on antioxidant enzyme catalase activity in H₂O₂-induced-stress human promyelocytic leukemia cells after 2 days

Figure 4 shows that there was no detection of IFN-γ in untreated HL-60 (no additives) and the group treated with only 50 μM H₂O₂. However, a significant enhancement of IFN-γ production was detected in the group of root extract treatment.

Figure 4.

Change in the levels of interferon-gamma-induced by human promyelocytic leukemia cells after treatment with 400 μg/mL Ligusticum porteri root extract for 2 days. ***This value is significantly different from other group treatments (P < 0.05)

Figure 5 shows that there was an increasing trend in IL-2 secretion when the cells were stress induced (50 μM H₂O₂) or treated with L. porteri root extract.

Figure 5.

Change in the levels of interleukin-2 induced by human promyelocytic leukemia cells after treatment with 400 μg/mL Ligusticum porteri root extract for 2 days. Figures that share different letters are significantly different (P < 0.05)

Figure 6 shows that hydrogen peroxide suppressed the production of IL-10. Treatment with 400 μg/mL of L. porteri root extract showed an increase of 63% in IL-10 levels, but this amount was still lower that the control, indicating that the root extract suppressed the inhibitory effect of H₂O₂.

Figure 6.

Change in the levels of interleukin-10 induced by human promyelocytic leukemia cells after treatment with 400 μg/mL Ligusticum porteri root extract for 2 days. Figures that share different letters are significantly different (P < 0.05)

DISCUSSION

The observed decline in HL-60 cells viability may be due to the inability of the medium to sustain the growth of the cells after 7 days of incubation. The enhancing survivability of HL-60 cells after treatment with L. porteri root extract may not be advantageous due to the malignant nature of the cells. Findings from this study do not support the application of L. porteri root extract as an antileukemic therapy. It is possible that the viability of HL-60 cells on day 0 was below the threshold of detection by the cell counting assay kit used. It is hypothesized that the addition of root extract to stressed HL-60 cells results in the suppression of the cell viability on day 0. It was previously reported that induction of 50 μM H₂O₂ for 4 h resulted in DNA fragmentation and triggered cell death in HL-60 cells.[27] Findings from this study showed that the treatment with root extract of L. porteri may protect HL-60 cells from oxidative stress caused by H₂O₂ similar to the effect observed in human peripheral blood lymphocytes.[17]

Lipid peroxidation is an important indicator of cellular damage caused by oxidative stress.[28] We have earlier reported the inhibition of lipid peroxidation, increased GSH, and increase in the activities of superoxide dismutase and catalase in human peripheral blood lymphocytes treated with L. porteri root extract.[17] In this study, the exposure of HL-60 cells to 50 μM H₂O₂ resulted in a significant increase in MDA content and depletion of GSH levels, which may be indicative of increased oxidative damage in these cells. However, we noted increase in GSH levels, SOD and CAT activities in stressed HL-60 supplemented with 400 μg/mL L. porteri root extract which is indicative of the protection of acute promyelocytic leukemia cells against oxidative stress.

To evaluate the immune-modulatory effects of the extract in HL-60 cells, we measured IL-2, IFN-γ and IL-10. We noted increase production of inflammatory mediators (IL-2 and IFN-γ) in stressed HL-60 cells treated with 400 μg/mL L. porteri root extract. We also noted significant up-regulation of IL-2 after a 2-day incubation with 400 μg/mL L. porteri root extract. Although the amount of IFN-γ was low compared to other interleukins (IL-2 and IL-10) in stressed cell cultures treated with 400 μg/mL L. porteri root extract, there was a significant increase in IFN-γ production when compared to the control. These findings suggest that the root extract of L. porteri has positive effect on oxidative stress and inflammation in acute promyelocytic leukemia cells.

CONCLUSION

Findings from this study do not support the use of L. porteri root extract as an anti-proliferation and differentiation therapy for acute promyelocytic leukemia cells. However, the results also showed that L. porteri root extract possess an antioxidation function and regulation on immune factors in HL-60 cells.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.