Crocin is a carotenoid of the saffron extract that exhibits antitumor activity against many human tumors. However, the effects of crocin on HL-60 cells in vivo have not been evaluated. This study aimed to examine the effects of crocin on HL-60 cells in vitro and in vivo and investigate the underlying mechanisms. HL-60 cells were treated by crocin, and cell proliferation, apoptosis, and cell cycle profiles were examined by MTT assay, AO/EB staining, and flow cytometry, respectively. Furthermore, HL-60 cells were xenografted into nude mice and treated by crocin, the tumor weight and size were calculated, and the expression of Bcl-2 and Bax in xenografts was detected by immunohistochemical staining. The results showed that crocin (0.625–5 mg/mL) inhibited HL-60 cell proliferation and induced apoptosis and cell cycle arrest at G0/G1 phase, in a concentration and time-dependent manner. In addition, crocin (6.25, 25 mg/kg) inhibited the tumor weight and size of HL-60 xenografts in nude mice, inhibited Bcl-2 expression, and increased Bax expression in xenografts. In summary, crocin inhibits the proliferation and tumorigenicity of HL-60 cells, which may be mediated by the induction of apoptosis and cell cycle arrest and the regulation of Bcl-2 and Bax expression.
Survival rates of children with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) currently range from 83% to 94% and 60% to 65%, respectively [
It has been reported that carotenoids from saffron were effective in inhibiting the proliferation of HL-60 cells [
Human leukemia HL-60 cells were gifted from the Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences, Tianjin. HL-60 cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS) in a humidified incubator of 5% CO2 at 37°C. Crocin was purchased from Sigma (CAS Number 42553-65-1) and diluted in 10 mmol/L phosphate-buffered saline for the appropriate concentration upon used.
Cell proliferation was determined by using MTT assay. Briefly, HL-60 cells were treated with crocin (0.625–10 mg/mL) for 24 h or 48 h. Then the cells were incubated with MTT solution (5 mg/mL in PBS, Sigma) for 4 h and solubilized with DMSO (150
AO/EB staining of HL-60 cells was performed to detect the apoptotic and necrotic patterns as described previously [
HL-60 cells were treated with different concentrations of crocin. After 48 h, cells were harvested and fixed in 70% ethanol at 4°C overnight. Fixed cells were stained with 5
A total of 32 males BALB/c nude mice (3 weeks old) were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Sciences. Animals were maintained under standardized, sterilized conditions (
Nude mice xenograft models were established by injecting HL-60 cells (
The immunohistochemical staining of Bcl-2 and Bax in the tumor tissue was performed using the streptavidin-biotin-complex peroxidase kit (Boster, Wuhan, China). Finally, the slides were washed, dehydrated, and mounted for microscopic examination and enumeration immunoreactive cells (yellow to brown). Analysis of immunostaining in xenografts was done on a Media Cybernetics Image-Pro Plus analysis system linked to an Olympus microscope. The cells stained positive for Bcl-2 and Bax were quantified by counting the yellow to brown cells and the total number of cells at five randomly selected fields at 400x magnification.
The tumor tissues were collected and lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitors. The protein concentrations of the lysate were quantified using the bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology, China). Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinyli-dene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked with PBST (PBS with 0.05% Tween-20) containing 5% nonfat dry milk for 1 h and then incubated at 4°C overnight with Bcl-2, Bax, or
Data were presented as the mean
MTT assay showed that compared with the control group, crocin at the various concentrations (0.625–10 mg/mL) significantly inhibited HL-60 cell proliferation, and the inhibitory effect of crocin on HL-60 cell proliferation was dose and time dependent (Figure
Crocin inhibits the proliferation of HL-60 cells in a dose-and time-dependent manner. HL-60 cells were treated by crocin at the indicated concentration for 24 or 48 h, and the inhibition rate of proliferation was calculated based on MTT assay. *
To determine whether crocin inhibits the proliferation of HL-60 cells through the regulation of cell cycle progression and apoptosis, first we performed flow cytometry using PI staining. We observed a significant increase of G0/G1 cells from 55.33% in control group to 70.27% in the crocin-treated group (5.0 mg/mL). However, at 10 mg/mL, crocin could not further increase the cell ratio in G0/G1 phase (Figure
Crocin induces apoptosis and cell cycle arrest of HL-60 cells. (a) HL-60 cells were treated by crocin at the indicated concentration for 48 h, and the ratio of cells at G0/G1 was calculated based on flow cytometry. (b) HL-60 cells were treated by crocin at the indicated concentration for 24 or 48 h, and the percentages of apoptotic cells were calculated based on AO/EB staining. *
AO/EB staining showed that uniformly green live cells with normal morphology were seen in the control HL-60 cells, whereas green early apoptotic cells with nuclear margination and chromatin condensation occurred in HL-60 cells treated by 0.625–2.5 mg/mL crocin, and orange later apoptotic cells with fragmented chromatin and apoptotic bodies were seen in HL-60 cells treated by 5 mg/mL. The percentage of apoptotic cell significantly increased gradually with crocin concentration increased from 0.625 to 5 mg/mL, compared with the control group, and the effects were time dependent (Figure
After being injected HL-60 cells, spontaneous activity and food intake of all mice decreased. At the time of receiving HL-60 cells, there was no significant difference in the body weight between the four groups. As the tumors grew, all the mice’s weight increased (Figure
The body weight of mice that received HL-60 xenografts and crocin treatment. The body weight of mice was monitored daily throughout the experiment. Left panel: the body weight of mice at the beginning of receiving xenografts. Right panel: the body weight of mice after 28 days of crocin treatment. *
The tumor formation rate of the control and experiment groups (6.25, 25, 100 mg/kg crocin) was 100%, 50%, 75%, and 75%, respectively. There was no significant difference in the tumor formation rate among the four groups. The tumor formation time of the control and the experiment groups was
At the end of the study, the xenografts were excised from each sacrificed mouse, and tumor weight and volume were calculated. Tumor weight and the change ratio of tumor size in mice treated by crocin at the doses of 6.25 and 25 mg/kg were both significantly inhibited compared with the control group (Figures
The tumor weight and size in mice that received HL-60 xenografts and crocin treatment. After 28 days of treatment, the mice were sacrificed, and the xenografts were excised. (a) Tumor weight in different treatment groups. (b) The change ratio of tumor size in different treatment groups. *
To investigate whether the regression of tumor growth by crocin is due to the induction of apoptosis in vivo, we performed immunohistochemistry analysis of Bcl-2 and Bax expression in xenograft. The number of Bcl-2 positive cells was decreased in tumors from mice treated by 6.25 or 25 mg/kg crocin, compared to those from controls. In contrast, the number of Bax positive cells was increased in tumors from mice treated by 6.25 or 25 mg/kg crocin, compared to those from controls (Figure
Immunohistochemical staining of Bcl-2 and Bax expression in HL-60 xenografts. (a) The percentages of cells stained positively for Bcl-2 and Bax in different groups. (b) Immunohistochemical staining of Bcl-2 in HL-60 xenografts from different groups. (c) Immunohistochemical staining of Bax in HL-60 xenografts from different groups. Magnification: 400x.
We also performed western blot analysis of Bcl-2 and Bax expression in xenografts. The results showed that the protein level of Bcl-2 was reduced in tumors derived from mice treated with 6.25 or 25 mg/kg crocin, compared to those from control. In contrast, the protein level of Bax was increased in tumors derived from mice treated with 6.25 or 25 mg/kg crocin, compared to those from control (Figure
Crocin regulates the expression levels of Bcl-2 and Bax in HL-60 xenografts. The mice were treated with crocin (0, 6.25, 25, or 100 mg/kg, qd), and xenografts were collected for western blot analysis. Left panel: representative blots. Right panel: quantization of relative Bcl-2 and Bax protein levels in different groups.
In the present study, we showed that crocin, a main compound derived from Crocus sativus extract, could inhibit the proliferation and induce the apoptosis of HL-60 cells both in vitro and in vivo. These data provide strong evidence that crocin has the potential for the treatment of leukemia.
Anti-tumor drugs are known to regulate cell cycle progression, inhibit cell growth and proliferation, and induce apoptosis in tumor cells [
To confirm our in vitro results, we employed nude mice xenograft model to evaluate the in vivo anti-tumor effects of crocin. Our results showed that crocin at the dose of 6.25, 25 mg/kg had strong inhibitory effect on HL-60 cell growth in nude mice, while the high dose (100 mg/kg) had no significant inhibitory effect, perhaps due to the toxic effects.
There was no accidental death throughout the course of the animal experiment, indicating the safety of crocin. It was demonstrated that orally administered crocin was not absorbed in plasma either after a single dose or repeated doses, and crocin was excreted largely through the intestinal tract following oral administration [
Medicinal herbs have been shown to exert anti-tumor effects by the induction of apoptosis in cancer cells including leukemia cells [
In summary, both in vitro and in vivo studies demonstrate that crocin inhibits the proliferation and tumorigenicity of HL-60 cells, which may be mediated by the induction of apoptosis and cell cycle arrest and the regulation of Bcl-2 and Bax expression. These findings suggest that crocin has the potential to be developed as a new drug with high efficacy and low toxicity for the treatment of leukemia.
The authors declare that they have no conflict of interests.