Pogostemon cablin extract as an anticancer agent on human acute myeloid leukemia

Abstract Pogostemon cablin has been indicated to treat many kinds of diseases and the progression of cancers, such as colorectal cancer. However, the effects of P. cablin extract (PPa extract) against acute myeloid leukemia have not been investigated. Thus, this study explored the anticancer potential of PPa extract and its mechanism in HL‐60 cells. The MTT assay results showed that PPa extract significantly inhibited the proliferation of HL‐60 cells in a dose‐dependent manner and affected cell morphology, causing cell shrinkage and the formation of debris. PPa extract blocked cell cycle progression at the G0/G1 phase in a dose‐ and time‐dependent manner and induced cell apoptosis, as shown by the observation of DNA fragments and apoptotic bodies. Furthermore, PPa extract caused the accumulation of a population of cells at G0/G1 phase via a reduction in p‐Rb, increasing p21 expression, and downregulating cell cycle regulator protein expression. Then, PPa extract was found to activate the extrinsic and intrinsic apoptosis pathways, leading to cell death. These data demonstrated that PPa extract exerted inhibitory activity and triggered cell apoptosis in HL‐60 cells and that PPa extract might be a chemopreventive agent for cancer therapy.


| INTRODUC TI ON
Natural products have been strongly investigated as promising anticancer agents. Numerous studies suggest that vegetables and fruits play a protective role in reducing the risk of cancer (Tsuda et al., (2004); Gullett et al., 2010). Pogostemon cablin is a traditional herbal medicine used in the treatment of many kinds of diseases, such as the common cold, diarrhea, headache, and fever (Lin et al., 2014). Moreover, its other effects, including its anti-inflammatory, antioxidant, antifungal, and antibacterial effects, have been widely reported (Kim et al., 2010Su et al., 2012;Vu et al., 2016;Lu et al., 2011;Kocevski et al., 2013).
Pogostemon cablin has been used as a complementary anticancer agent in colorectal cancer (Tsai et al., 2015). However, the mechanisms underlying the anticancer activity of P. cablin extract (PPa extract) in acute myeloid leukemia cells have yet to be understood. Acute myeloid leukemia (AML) is a hematological disease, and its incidence rate is 15%-20% in those who aged 15 years or younger and 80% in the patients above the age of 65 years (Deschler & Lübbert, 2006). The present chemotherapeutic approach for AML is the eradication of leukemia cells with a minimal reduction in normal cells, which has significantly improved the rate of remission. However, the relapse rate is more than 50%, with subsequent resistance leading to the death of most AML patients (Lagunas-Rangel et al., 2017).
In recent years, the concept of cancer prevention has gained much attention, and the induction of apoptosis and cell cycle regulation have been suggested as targets in cancer treatment. Apoptosis, a process of programmed cell death in which cells are eliminated without the release of harmful substances, is essential for development and homeostasis. Moreover, it is activated by a serial caspase cascade including caspase-8, caspase-9, and caspase-3, leading to cell apoptosis (Lopez & Tait, 2015;Koff et al., 2015;Baig et al., 2016;Goldar et al., 2015;Ichim & Tait, 2016). Cells progress through the cell cycle during cell proliferation, during which cell growth is controlled via cell cycle regulators and tumor suppressors, such as p53 and Rb (Jung et al., 2010;Giacinti & Giordano, 2006;Abbas & Dutta, 2009). Many studies have indicated that cancerous cells often present dysfunctional cell cycle regulation, causing uncontrolled cell proliferation. Thus, the purpose of this study was to investigate the antileukemic activity of PPa extract on apoptosis and cell cycle regulation in vitro. Jurkat cells were cultured in RPMI 1640 medium. The media were supplied with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% sodium pyruvate. Cells were incubated at 37°C in a humidified incubator containing 5% CO 2 . All other cell culture reagents were purchased from Gibco/Thermo Fisher Scientific, and all other chemicals were of research grade. HL-60 and the FemtoPath TP53 exon 8 primer set was used to confirm the TP53 levels in K562 cells. The FemtoPath KRAS exon 2 primer set, FemtoPath EGFR exon 19 primer set, and FemtoPath PIK3CA exon 9 primer set (HongJing Biotech, New Taipei City, Taiwan) were used to confirm the EGFR, KRAS, and PIK3CA levels, respectively, in K562 cells.

| Preparation of Pogostemon cablin extract
Fresh leaves of Pogostemon cablin plant from Indonesia were carried out with steam distillation and had been testified in our laboratory in small scale. Plant material (400 g) was penetrated with generated steam with 7.2 ml/min of flow rate at 100 ~ 105℃ for 100 min and had commissioned by Phoenix in large scale. PPa extract which was obtained from lipid layer was dissolved in DMSO to determine the concentration in μg/ml by the equation: 20 μl of PPa extract (g)/(180 μl of DMSO + 20 μl of PPa extract) (g). After that, the growth medium was utilized to dilute the PPa extract and the final concentration of DMSO in cells was less than 1%.

| Cell viability assay
The effect of PPa extract on cell viability was determined by the MTS assay. The PPa extract was produced by steam distillation and obtained from PHOENIX. Cells (5 × 10 3 /50 μl) were grown in a 96-well plate for 3-6 hr and then incubated with PPa at a series of concentrations for 12, 24, and 48 hr. After the treatment period, MTS was added to each well, and cells were further incubated for 12 hr. The absorbance at 490 nm was measured using a SpectraMax Plus 384 microplate reader (Molecular Devices). Cell viability is expressed as a percentage of the value for a control culture, which was considered 100% viable.

| Flow cytometric analysis of the cell cycle
HL-60 cells were plated at a density of 5 × 10 6 cells/well in 10-cm dishes in the presence of 0, 5, 15, and 25 μg/ml PPa extract for the indicated time intervals. The collected cells were stained with PI in the presence of RNase A and incubated at 4°C overnight. The DNA content was analyzed using a flow cytometer (BD) equipped with CellQuest Pro software.

| TUNEL staining
HL-60 cells were incubated with PPa extract (15 μg/ml) for 24 hr, and the cells were then gently smeared on slides to study apoptotic induction. The cells were fixed with 10% formalin in the presence of methanol, followed by detection with an in situ cell death detection kit (Roche, Germany) according to the manufacturer's instructions.
After incubation, the slides were washed and immediately visualized under a fluorescence microscope (ZEISS Axioskop2, USA) at 400× magnification to detect apoptotic cells.

| Total protein extraction and Western blot analysis
HL-60 cells were treated with PPa extract (0, 5, 15, and 25 μg/ml) for 6, 12, 24, and 48 hr. To extract the total protein, the collected cells were lysed with lysis buffer. Cell lysates were separated by SDS-PAGE, and proteins were transferred to PVDF membranes. After membrane blocking, the membranes were incubated with primary antibodies at 4°C overnight, followed by incubation with HRP-conjugated secondary antibodies. Subsequently, the blots were detected using the T-Pro LumiFast Plus chemiluminescence detection kit (T-Pro Biotechnology, USA), and signals were captured using an Image Quant LAS 4000 image reader (GE Healthcare Life Sciences). Primary antibodies against p-Rb, p21, PCNA, cdk2, cdk4, cyclin B1, cyclin D1, FAS, bax, VEGF, MMP2, MMP9, caspase-3, caspase-8, and caspase-9, and horseradish peroxidase (HPR)-conjugated secondary antibody were purchased from Santa Cruz Biotechnology, Inc. and iReal Biotechnology Co., Ltd.
These data were performed three independently experiments. Center for Advanced Instrumentation. The sample was diluted using hexane (1/500). The oven temperature was programmed to 300⁰C/1 ml/min. Helium was used as carrier gas at flow 1.0 ml/min. The identification of compounds was based on comparison of their mass spectra with those of WILEY and NIST Libraries.

| Statistical analysis
The results are presented as the mean ± standard deviation (SD).

Statistical analyses between various groups were performed by
Student's t test. Significance was indicated by p < .05 in all experiments.

| PPa extract inhibited the growth of leukemia cells
The inhibitory effect of PPa extract against HL-60, RAW 264.7, and P338D1 cells was measured by MTS assay. The cells were treated with PPa extract at a series of concentrations for 12, 24, and 48 hr, as shown in Figure 1. PPa extract significantly inhibited the growth of these cells in a dose-dependent manner. For HL-60 cells, treatment with PPa extract at a lower concentration (<25 μg/ml) sharply decreased cell viability, and treatment with PPa at concentrations ranging from 50 to 200 μg/ ml had a marked inhibitory effect. Furthermore, HL-60 cells were more sensitive to PPa extract treatment for 48 hr compared to other periods.
However, treatment with PPa extract at a low concentration (<25 μg/ ml) for 12, 24, and 48 hr significantly increased the proliferation of RAW 264.7 and P338D1 cells. As a result, PPa extract showed the ability to induce normal macrophage proliferation instead of an inhibitory effect on normal macrophages; in contrast, PPa extract had a highly efficient inhibitory effect on HL-60 cells. To further examine the inhibitory effect of PPa extract on the proliferation of different types of leukemia cells, Jurkat (Acute lymphocytic leukemia, ALL) and K562 (Chronic myeloid leukemia, CML) cells were also investigated by MTS assay, as shown in These results revealed that PPa extract exerts a stronger inhibitory effect on leukemia cells in a dose-dependent manner and lower cytotoxicity against normal macrophages; moreover, PPa extract showed the ability to induce macrophage proliferation.

| PPa extract-induced cell cycle arrest in HL-60 cells
To elucidate the mechanisms underlying the inhibitory effect of PPa extract on cell growth, the cell cycle distribution of HL-60 cells was analyzed by flow cytometry. HL-60 cells were treated with PPa extract (5, 15, and 25 μg/ml) for 24 hr, and the population of cells in G 0 /G 1 phase significantly increased from 48.3% to 68.1%, accompanied by a decrease in the population of cells in S phase from 13.8% to 4.4% and in the population of cells in G 2 /M phase from 31.4% to 11.6%. Similarly, after PPa extract treatment (15 μg/ml) for 6, 12, 24, and 48 hr, the accumulation of cells in G 0 /G 1 phase was markedly increased, followed by a decrease in the percentage of cells in the S and G 2 /M phases, as shown in Table 2. These results indicate that PPa extract can induce cell cycle arrest at G 0 /G 1 phase in a dose-and timedependent manner, as shown in Figure 2. Furthermore, these results suggest that the inhibitory effect of PPa extract on HL-60 cell is due to the induction of cell cycle arrest at G 0 /G 1 phase.

| PPa extract-induced apoptosis in HL-60 cells
The results of cell cycle distribution analysis also revealed that the percentage of cells in the sub-G 1 phase was increased after PPa extract treatment in a dose-dependent manner (Figure 3a). Moreover, microscopic examination revealed that HL-60 cells exposed to PPa extract (5, 15, and 25 μg/ml) exhibited cell swelling, shrinking, and debris, indicating that PPa extract contributed to HL-60 cell death ( Figure 3b). To further detect the ability of PPa to induce apoptosis, HL-60 cells were incubated with PPa extract (15 μg/ml) for 24 hr and subjected to a TUNEL assay, followed by PI staining. As shown in Figure 3c

| Effects of PPa extract on the cell cycle and cell apoptosis
To All experiments were independently performed three times, and representative results are shown. Statistically significant differences are indicated with *p < .05 F I G U R E 4 PPa extract induced cell cycle regulation and apoptosis activation. HL-60 cells were treated with PPa extract at different concentrations for the indicated periods. Protein expression with or without PPa extract treatment was determined by Western blot analysis (GAPDH was used as a loading control.). The experiment was repeated three times, and representative blots are shown the expression of p-Rb, whereas the expression of p21 was upregulated in a drug concentration manner with PPa extract treatment.

| D ISCUSS I ON
Natural plants have been widely used to treat many kinds of diseases for centuries and are also used as a daily medicine or functional food for the prevention of disease. With the incidence of cancer, preventative medicine has received more attention worldwide and has been rapidly expanding, even in developed countries, in recent years. For instance, traditional herbal medicine accounts for 30%-50% of the total medicinal consumption in China (Tsuda et al., 2004;Gullett et al., 2010).
P. cablin is an herbal plant that has been used widely to treat fever and inflammation and to alleviate pain (Lin et al., 2014). Azulene has been reported the inhibitory ability on breast cancer cells (MCF-7) and prostate cancer cells (DU145) (Ayaz et al., 2020).
In conclusion, P. cablin extract induced cell cycle arrest and apoptosis, which might have become new therapeutic targets in human myeloid leukemia cells for cancer treatment. These results provide new insights into the possible molecular machinery underlying the activity of PPa extract, indicating its potential as a promising chemopreventive agent for cancer treatment or as a daily preventive medicine. However, further investigation of the components of PPa extract responsible for its efficacy will be important for the application of PPa extract.

ACK N OWLED G M ENTS
The work was also supported in part by Grants TCCRD106-20 from