Study of the antitumor mechanisms of apiole derivatives (AP-02) from Petroselinum crispum through induction of G0/G1 phase cell cycle arrest in human COLO 205 cancer cells

Background Apiole was isolated from the leaves of various plants and vegetables and has been demonstrated to inhibit human colon cancer cell (COLO 205 cells) growth through induction of G0/G1 cell cycle arrest and apoptotic cell death. This study further explored the antitumor effects of apiole derivatives AP-02, 04, and 05 in COLO 205 cancer cells. Methods Human breast (MDA-MB-231, ZR75), lung (A549, PE089), colon (COLO 205, HT 29), and hepatocellular (Hep G2, Hep 3B) cancer cells were treated with apiole and its derivatives in a dose-dependent manner. Flow cytometry analysis was subsequently performed to determine the mechanism of AP-02-induced G0/G1 cell cycle arrest. The in vivo antitumor effect of AP-02 (1 and 5 mg/kg, administered twice per week) was examined by treating athymic nude mice bearing COLO 205 tumor xenografts. The molecular mechanisms of AP-02-induced antitumor effects were determined using western blot analysis. Results AP-02 was the most effective compound, especially for inhibition of COLO 205 colon cancer cell growth. The cytotoxicity of AP-02 in normal colon epithelial (FHC) cells was significantly lower than that in other normal cells derived from the breast, lung or liver. Flow cytometry analysis indicated that AP-02-induced G0/G1 cell cycle arrest in COLO 205 cells but not in HT 29 cells (< 5 μM for 24 h, **p < 0.01). Tumor growth volume was also significantly inhibited in AP-02 (> 1 mg/kg)-treated athymic nude mice bearing COLO 205 tumor xenografts compared to control mice (*p < 0.05). Furthermore, G0/G1 phase regulatory proteins (p53 and p21/Cip1) and an invasion suppressor protein (E-cadherin) were significantly upregulated, while cyclin D1 was significantly downregulated, in AP-02-treated tumor tissues compared to the control group (> 1 mg/kg, *p < 0.05). Conclusions Our results provide in vitro and in vivo molecular evidence of AP-02-induced anti-proliferative effects on colon cancer, indicating that this compound might have potential clinical applications. Electronic supplementary material The online version of this article (10.1186/s12906-019-2590-9) contains supplementary material, which is available to authorized users.


Background
Colon cancer is the second most common cause of cancer-related death following lung cancer [1,2]. However, clinical therapeutic approaches for treating colon cancer are still limited to surgical resection, radiation and chemotherapy [3][4][5]. For that reason, scientists have continued to investigate additional targeted therapeutic strategies [6]. The present study was based on these principles and aimed to identify small molecules that specifically induce cell cycle arrest at G0/G1 phase and trigger the cellular apoptotic response in cancerous cells.
Apiole, 1-allyl-2,5-dimethoxy-3,4-methylenedioxybenzene, also referred to as parsley apiol or simply apiol, is a phenylpropene derivative purified from various natural sources, such as the fruits of Petroselinum crisp [7], the seeds of Enterolobium contortisiliquum (leguminosae) [8], wild-growing Salvia aegyptiaca [9], and the leaves of Cinnamomum verum Presl [10], caraway (Carum carvi L.) [11] and Pituranthos chloranthus ssp. [12]. Medicinally, it has been used to treat menstrual disorders and as an abortifacient in ancient times. We previously studied the molecular mechanisms of apiole-induced G0/G1 cell cycle arrest and subsequent induction of apoptosis specifically observed in human colon cancer cells (COLO 205 cells) [13]. G0/G1 cell cycle arrest-related proteins, such as p53, p21 and p27, were upregulated, and the cyclin D1 protein was downregulated. In addition, we demonstrated that apoptotic cell death was induced by apiole through activation of caspase (caspase 3,8,9)-mediated pathways. We found that bax/bcl-2 triggering signals were activated with significantly induced DNA laddering formation and induction of a subG1 peak observed by flow cytometry analysis. While the detailed mechanism of the apiole-induced anti-proliferative effects in COLO 205 cells requires further investigation, our findings clearly indicate that apiole is a candidate compound for the development of clinical anticancer drugs.
Considering the aforementioned findings, along with the crucial goal of identifying more specific anti-cancer drugs, three apiole derivatives (AP-02, AP-04, and AP-05) were either chemically synthesized or commercially procured and subsequently evaluated for their anti-proliferative activity.
Cell synchronization, drug treatments, and flow cytometry analysis Cells were washed with PBS three times 24 h after plating and incubated with media containing 0.04% fetal calf serum (FCS) for an additional 24 h. Under these conditions, cells are arrested in G0/G1 phase based on flow cytometry analysis as reported in our previous study [18]. Next, synchronized cells (cultured in 0.04% FCS) were challenged with the addition of media containing 10% FCS. Apiole-and PBS-treated groups were assessed via flow cytometry analysis to determine cell cycle distribution. Cells were stained with propidium iodide (50 μg/ml) (Sigma Chemical Co., St. Louis, MO, USA), and DNA content was assessed using a FACScan laser flow cytometry analysis system (Becton-Dickinson, San Jose, CA, USA), with 15,000 events being analyzed for each sample.

Animals
Four-week-old athymic nude mice were purchased from the National Science Council Animal Center, Taipei, Taiwan. Five animals in each cage were fed and acclimatized in our experimental animal center for 2 weeks in rectangular cages, and a small wood strip served as environmental enrichment. Animals were fed with the lab diet 5 k52 formulation (6% fat), and water was accessible at all times. Our animal facility was maintained under standard specific-pathogen-free conditions and a 12 h/ 12 h light/dark cycle. The total number of animals used in this experiment was 21, and the animals were equally divided among the 3 groups based on tumor volume. Sample size calculation was considered as follows: effect size = total number of animals − total number of groups, and the number of animals in each experimental group was n = 7. If the tumor size was greater than 4 cm 3 or the mouse weight was 15% below the original weight, euthanasia was performed by placing the mice in a chamber and piping in carbon dioxide (CO 2 ) at increasing concentrations until the animals became unconscious and died.

Treatment of COLO 205-derived xenografts in vivo
COLO 205 cells were cultured in media as described above. Cells (5 × 10 6 ) were suspended in 0.2 ml medium and injected subcutaneously between the scapulae of nude mice (purchased from National Science Council Animal Center, Taipei, Taiwan). For determination of tumor growth, the tumor volume was measured according to the following formula: tumor volume (mm 3 ) = L x W 2 /2, where L is the length and W is the width [19]. Once tumors reached a mean size of 200 mm 3 , experimental animals were treated with either 25 μl PBS or 1 to 5 mg/kg AP-02 via intraperitoneal injection three times per week for 6 weeks. Control animals were treated with PBS at the same volume.

Statistical analysis
For each analysis, data are represented as the mean ± SEM of at least three independent experiments. For comparison, statistical significance was tested using ttests. All p-values were based on two-sided statistical analyses, and p < 0.05 was considered statistically significant.
Low concentrations of Ap-02 inhibit human colon cancer cell proliferation through induction of G0/G1 cell cycle arrest Based on the results described above, we next investigated the specific mechanisms of AP-02 against cancer cells and selected COLO 205 cells as an in vitro cell model to test for cell cycle inhibition effects using flow cytometry (Fig. 5). We synchronized COLO 205 cells by culturing them in 0.04% FCS medium for 24 h according to our previous studies [18,20]. Synchronized cells were than treated with 10% FCS to reactivate cell proliferation (Fig. 5, red bars). Our previous studies [18,20] showed that the major differences in G0/G1 cell populations between serum starved and reactivated (10% FCS-treated) COLO 205 cells were observed 15 h after the media was replenished with complete media (Fig. 5a). COLO 205 cells in the PBS-treated group were in S phase at this time point (15 h) [20,21]. According to these data, we selected this time point (15 h) to test AP02-induced G0/ G1 arrest effects across a range of doses. The minimal dose of AP-02 that induced G0/G1 arrest was evaluated via flow cytometry analysis (Fig. 5b). Results indicated that the minimal dose of AP-02 needed to induce significant G0/G1 arrest was 5 μΜ (Fig. 5b, green vs. red bars, *p < 0.05). We further demonstrated that higher doses of AP-02 (> 5 μM for 24 h) induced appearance of a significant subG1 phase cell population (Fig. 5b, yellow and blue bars, *p < 0.05). These results indicate that AP-02-induced cancer cell death was occurring in high-dose groups.

Ap-02 induces antitumor effects in vivo in COLO 205xenograft tumors
To evaluate AP-02-induced antitumor effects, we used athymic nude mice bearing COLO 205 tumor xenografts  (Fig. 6). No gross signs of AP-02-induced drug toxicity were detected in body weight changes (Fig. 6b). The general and microscopic appearance of individual organ tissues was also assessed (data not shown).

G0/G1 cell cycle and metastasis regulatory proteins are involved in Ap-02-induced antitumor effects in COLO 205xenograft tumors
In this study, we found that high doses (> 25 μΜ) of AP-02 induced the appearance of a significant subG1 phase cell population in COLO 205 cells compared to the 10% FCS-treated group (Fig. 5b, *p < 0.05). However, in the lower dose Apo2-treated group (< 5 μΜ), only G0/G1 cell cycle arrest was detected (Fig. 5b, green bar, *p < 0.05). Based on these results, we suggest that G0/G1 cell cycle regulatory proteins are likely involved in AP-02-induced effects. We next confirmed these in vivo observations in AP-02-treated (1 and 5 mg/mL) COLO 205-xenograft tumor tissues. We found that expression of p53 and p21/Cip1 proteins was significantly induced, while cyclin D1 was downregulated in AP-02-treated tumor tissues (> 1 mg/kg) compared to controls (Fig. 7). To test whether AP-02 effectively inhibited metastasis-related signals in COLO 205-xenograft tumor cells, we selected Ecadherin, which functions as an invasion suppressor, as an indicator and determined whether it was upregulated in AP-02-treated tumors. We demonstrated that AP-02 treatment upregulates expression of E-cadherin protein (> 1 mg/kg) (Fig. 7). These results suggest that induction of E-cadherin by AP-02 inhibits COLO 205 cancer cell migration. The E-cadherin protein has also been used as a target for therapeutic purposes to prevent colorectal cancer cell metastasis [22]. Therefore, AP-02 may have clinical utility as a novel small molecule to prevent colorectal cancer cell metastasis.

Discussion
Petroselinum crispum has been used as a medicinal herb. Plant extracts isolated from either the leaf or stem have been evaluated for antioxidant and DNA damage protection effects in normal (3 T3-L1) cells, in addition to their antitumor effects in breast cancer (MCF-7) cells [23]. Plant extracts from Petroselinum crispum have been investigated for different medicinal properties, including anti-mosquito properties [24] and anti-cadmium neurotoxicity in albino mice [25]. Toxicity and hematological studies in rats indicated that Petroselinum crispum was nontoxic at the applied dosage (< 1000 mg/kg) [26]. In this study, the antitumor effects of AP-02 derived from Petroselinum crispum were tested in human colon cancer cells. Interestingly, in vitro studies demonstrated that AP-02 administration induced significant G0/G1 cell cycle arrest in COLO 205 cells at concentrations less than 5 μΜ compared to the 10% FCS-treated group. However, this effect was not observed in normal human colon epithelial (FHC) cells. To examine the mechanism whereby these effects occur, an in vivo study was performed, which indicated that AP-02 affects G0/G1 phase regulatory proteins, significantly inducing p53 and p21/ Cip1 protein expression and downregulating cyclin D1 expression compared to the control-treated group. Taken together, these results demonstrate that AP-02 may have potential for antitumor application through induction of cell cycle inhibition, specifically G0/G1 phase arrest, in colon cancer cells. According to previous studies, apiole has been isolated from many plants [8,27]. For example, Petroselinum crispum [27], which is used as a culinary herb and widely used as a seasoning condiment, is in the Apiaceae family. In this study, the antitumor effects of apiole derivatives (AP) were tested in different human cancer cell lines. Interestingly, human COLO 205 colon cancer cells were the most sensitive cells in responding to Ap-02 treatment. Another study further demonstrated that Petroselinum crispum extract induces cytotoxicity in hepatocellular carcinoma (Hep G2) cells [28]. Another study demonstrated that extract isolated from the seed of Enterolobium contortisiliquum exerts anti-migratory effects on gastric cancer cells [29,30]. Extract from the leaves of Cinnamomum verum Presl [8] demonstrated antitumor effects in human colon cancer cell lines through targeting topoisomerase 1 and 2 [31,32]. These studies all reveal the prominent antitumor activity of these plant extracts. However, the components mediating these effects were not identified. Our study provides evidence indicating that apiole derivatives, which were detectable in these plant extracts, may have antitumor properties.
Interestingly, we found that AP-02 was the most effective at inhibiting COLO 205 cell proliferation compared to another colon cancer cell line, HT 29. We suggest that p53-mediated p21 upregulation is likely the major mechanism involved in AP-02-induced G0/ G1 phase cell cycle arrest. The p53 protein is well known for induction of cancer cell growth cycle arrest, as well as for inducing apoptotic cell death [33]. Wild type p53-mediated p21/CIP1-activated G0/G1 arrest has been implicated as a major anti-proliferation factor in cancer cell responses to antitumor drugs [33,34]. Similar results were observed in a previous study, which indicated that rosiglitazone, a clinically used anti-type-2 diabetes drug, reduced tumor metastasis in human cancer cells. The study demonstrated that rosiglitazone had a significantly increased cytotoxic effect in p53-wild-type HCT116 cells but not in mutant p53 HT-29 cells [35]. Inhibition of MDM2, a negative regulator of p53, in addition to MDMX, which stabilizes p53, has been revealed as an effective strategy for improving radiotherapy outcomes [36]. For clinical radiotherapy, validation of wild type p53 protein expression in cancer cells is suggested. A similar study demonstrated that upregulation of wild type p53 effectively activates an early-onset breast cancer-related gene (GAS7) and, through the GAS7-CYFIP1-mediated signaling pathway, effectively suppresses breast cancer metastasis [37]. These results suggest that natural compounds, such as AP-02, that inhibit early-stage tumor cell formation through the activation of wild type p53-mediated signals might be valuable for chemoprevention. According to our previous study, we demonstrated that 4,7-Dimethoxy-5-methyl-l,3-benzodioxole (SY-1) which was isolated from fruiting body of Antrodia camphorate induced significant cell cycle arrest (50-150 μM) and apoptosis (> 150 μM) in colon cancer cells [38,39]. The SY-1 derivative Apiole is also present in different types of natural plant products [40]. We further demonstrated that Apiole is a potential antitumor agent tested by in vivo animal study [13]. To test the dosage range of Apiole, we examined the therapeutic efficacy of apiole in vivo (1, 5, or 30 mg/Kg body weight respectively) 3 times per week for 30 days by treating athymic mice bearing COLO 205 tumor xenografts. No gross signs of toxicity were observed in the mice receiving the treatment regimens.
After 30 days, the tumor growth in the groups treated with doses higher than the 1 mg/Kg body weight apiole was significantly inhibited relative to the growth observed in the control-treated mice. In this study, the Apiole derivatives (AP-02) were tested for effective in vitro cytotoxicity (5 μM) and in vivo antitumor activity compared to the control group (> 1 mg/kg, *p < 0.05). The dosage is lower than the Apiole as described. These results may have relevance for colon cancer chemotherapy.
Our finding suggest that AP-02 inhibits cell cycle progression, which is regulated by successive, coordinated activation of CDKs [41]. We also found that AP-02 affects dysregulation of a series of regulatory subunits called cyclins and a group of CDK-inhibitory proteins (CKIs) [42,43]. Among these CKIs, the two most well known are p21/Cip1 and p27/Kip1. This study demonstrated that AP-02 effectively induces p21/Cip1and p53 protein upregulation in COLO 205 xenograft tumors in vivo. p21/Cip1 expression may be induced by the p53 tumor suppressor gene in human cancer cells [44,45]. In addition, an in vivo study further demonstrated that cyclin D1 protein levels were downregulated. These results suggest that activation of p21/Cip1 by AP-02 is associated with and inactivates CDKs, resulting in cell cycle arrest [43,46]. In addition, E-cadherin has been reported to function as an invasion suppressor and is downregulated in most carcinomas. In contrast, N-cadherin is a reported invasion promoter that is upregulated in proliferative cancer cells. Upregulation of N-cadherin in epithelial cells induces morphological changes, including conversion to a fibroblastic phenotype, leading to malignant cancer cell phenotypes with motility and invasive characteristics. In our study, we found that after AP-02 treatment, E-cadherin protein was significantly upregulated ( Fig. 7) compared to the control-treated group. These results suggest that AP-02 is a potential therapeutic agent for the prevention of tumor cell metastasis.

Conclusion
Our study reveals the detailed mechanisms underlying AP-02-induced antitumor effect on COLO 205 xenograft tumor growth formation. Our experimental findings demonstrate that AP-02 inhibits cancer cell proliferation through G0/G1 cell cycle arrest (< 5 μM, low dose) in addition to apoptosis induction (high dose) in wild-type p53 colon cancer (COLO 205) cells, both in cultured cells and in xenograft animals. These results highlight the molecular mechanism by which AP-02 induces antitumor effects in colon cancer, which might have clinical relevance for future applications. Tumor tissues were dissected to isolate protein lysate extracts. Protein extracts (100 μg per lane) were separated via SDS-PAGE. After electrophoresis, proteins were transferred onto Immobilon-P membranes, which were probed with the proper dilution of specific antibodies. Proteins were then detected using the NBT/BCIP system. Membranes were also probed with anti-GAPDH antibody to correct for differences in protein loading. M, molecular weight marker. Representative data from two of seven tested animals are shown. The number below each line was detected by densitometry analysis and indicates the relative intensity of protein expression compared to the control-treated group (defined as 1 in mouse #1)