Umbelliprenin induces autophagy and apoptosis while inhibits cancer cell stemness in pancreatic cancer cells

Abstract Background Umbelliprenin is a sesquiterpene coumarin isolated from Artemisia absinthium L. and shows antitumor effects in various cancers by inducing apoptosis. However, the antitumor effect of umbelliprenin in human pancreatic cancer has not been clarified. Methods The antitumor effects were determined by MTT and AnnexinV/PI double staining assay in vitro and xenograft mice in vivo. Autophagy was determined via immunofluorescence analysis. Apoptotic or autophagic related proteins were measured by immunoblotting. The pancreatic cancer cell stemness were determined by mammosphere formation and ALDEFLUOR assay. Results It revealed that umbelliprenin inhibited pancreatic cancer cell proliferation in vitro and pancreatic cancer tumor growth in vivo. Moreover, umbelliprenin induced pancreatic cancer cell BxPC3 apoptosis and autophagy as evidenced by upregulated apoptosis and autophagy‐ related protein expression (p < 0.01). Blocking autophagy by 3‐MA or Atg7 knockout enhanced umbelliprenin‐induced apoptosis (p < 0.05). Umbelliprenin also reduced pancreatic cancer cell stemness by reducing Oct4, Nanog, and Sox2 mRNA levels (p < 0.01). Mechanistically, umbelliprenin greatly inhibited Akt/mTOR and Notch1 signal pathway. Conclusion Umbelliprenin may be a novel therapeutic approach for pancreatic cancer treatment.

However, other treatment modalities are extremely lacking. Therefore, the development of novel anti-pancreatic cancer agents with fewer side effects are urgently needed.
Pancreatic cancer contains various types of cells including a distinct subpopulation of pancreatic cancer stem cells (CSCs). Pancreatic CSCs contribute to tumor cell selfrenewal, differentiation, cancer recurrence, metastasis, and therapy resistance. 4,5 Markers that used to identify pancreatic CSCs include SOX2, OCT4, NANOG, ALDH1, CD24, CD44, and ESA. 6,7 Pancreatic CSCs are regulated by aberrant activation of signal pathways like NOTCH, WNT, and βcatenin. 8,9 Therefore, targeting these signal pathways may provide new therapeutic strategies in the treatment of pancreatic cancer.
In recent years, medicinal plants have grabbed great attention for treating diseases such as cancer. 10,11 Artemisia absinthium L. (Chinese name: Zhong Ya Ku Hao), is widely distributed in Asia, Europe, and North America and used as folk medicine to treat anorexia, parasitic diseases, bacterial infection, and cancer. [12][13][14] Additionally, A. absinthium extracts have been reported to inhibit breast cancer cell proliferation. 15,16 The methanolic extract of A. absinthium was reported to induce DNA damage and apoptosis in human colon cancer cells. 17 Umbelliprenin is a 7-prenyloxycoumarin extracted from A. absinthium, which shows antitumor effects in various cancer cell lines. Umbelliprenin inhibited human melanoma cell proliferation by inducing apoptosis and cell-cycle arrest. 18 In addition, umbelliprenin can inhibit cell migration and invasion in prostate cancer, gastric cancer, colorectal cancer, ovary cancer, and lung cancer. 15,16,[18][19][20][21] However, the anticancer effect of umbelliprenin in pancreatic cancer have not been clarified yet. Autophagy is a tightly orchestrated self-digestion process that sequesters misfolded proteins and damaged organelles into autophagosomes and fuses with lysosomes for degradation. 22 Serval upstream regulators like Akt/ mTOR, AMPK, and ROS can directly mediate autophagosome formation. 23 Upregulated autophagy has been reported to promote tumor growth and help tumor cell survival from various stresses like starvation, radiotherapy, chemotherapy, or other anticancer agents. However, whether umbelliprenin could trigger autophagy in pancreatic cancer and the underlying mechanisms are still not clearly determined.
In the current study, we examined the anticancer role of umbelliprenin in pancreatic cancer and investigated its underlying mechanisms involving Akt/mTOR and Notch1 signaling pathways to regulate apoptosis, autophagy, and cancer cell stemness.
For MTT assay, 1 × 10 4 exponentially growing Capan-1, BxPC3, and PANC-1 cells were seeded into 96-well culture plates overnight. The medium was then exchanged with DMEM containing different concentrations of umbelliprenin (10,20,40,80, and 160 μg/mL) and DMSO groups as a control for 24 or 48 h. After that, MTT assay was conducted by using MTT in accordance with the manufacturer's instructions and measured at 540 nm with a microplate reader (Thermo Fisher Scientific, Inc.).

| Western blot
For western blot assay, BXPC-3 cells were plated in 6 cm dishes(1 × 10 6 cells/dish) and treated with different concentrations of umbelliprenin or 3-MA as indicated. After incubation for 24 h, cells were harvested and extracted by RIPA buffer lysis buffer on ice (Beyotime Institute of Biotechnology). Cell lysate was centrifuged at 14,000 rpm for 10 min and the supernatants were collected. The protein concentrations were quantified by the Quick Start Bradford Dye Reagent (Bio-Rad). Total protein was mixed by adding 6× loading buffer and boiled for 5 min. A total of 30 μg proteins were measured by 4%-12% SDS-PAGE (Amersham Bioscience). Following incubated with the primary and secondary antibodies, the membranes were detected by ECL (Thermo Scientific) and analyzed on a ChemiDoc XRS+(Bio-Rad Laboratories, Inc.).

Immunofluorescence microscopy
BxPC3 cells were transfected with TF-LC3 (mRFP-GFPtandem fluorescent LC3) plasmid (#21074, addgene) by using lipofectamine 3000 (Invitrogen) and seeded on coverslips at 10000/well before receiving umbelliprenin or DMSO treatment. After added umbelliprenin or DMSO for 24 h, the cells were washed with PBS and fixed with 4% paraformaldehyde and stained with DAPI (ab228549, abcam). The green and red puncta were imaged by Zeiss LSM 700 confocal microscope equipped with a 63× oil immersion objective (Zeiss GmbH).

| Immunoprecipitation (IP) assays
Following culture in the 6 cm plates for 24 h, BxPC3 cells were transiently transfected with 4 μg of pCDNA4-Vps34-Flag (#24398, addgene) plasmid with Lipofectamine 3000 (Invitrogen) and then treated with umbelliprenin or DMSO. The cell lysates containing 500 μg of total protein were mixed with 1 μg of Vps34 antibody and protein A/G beads overnight. The samples were separated by SDS-PAGE and visualized with enhanced ECL reagents.

| Xenograft model
The animal experiments and protocol were approved by the animal ethics committee of Shanghai Jiao Tong University (2021-08). A total of 20 nude mice (BALB/c; age, 4-6 weeks; weight, 18-20 g; male) were purchased from Shanghai Laboratory Animal Center and were fed in a specific pathogen-free (SPF) room. To determine the in vivo anticancer efficacy of umbelliprenin, BxPC3 cells (4 × 10 6 cells) were injected subcutaneously into the flank of each nude mouse. When the tumor volume reached about 100 mm 3 , the nude mice were randomly divided into five groups for different treatments (each has four mice): (i) control group (DMSO dissolved in PBS, once daily i.p.); (ii) 3-MA group (25 mg/kg/day, i.p.); (iii) umbelliprenin group (50 mg/kg/day, i.p.); (iv) gemcitabine group (100 mg/kg, twice weekly, i.p.) and (v) umbelliprenin(50 mg/kg) +3-MA (25 mg/kg) group (once daily, i.p.). 25 The body weight and tumor volume were measured every 4 days. 28 days after transplanting, the mice were sacrificed and the tumor samples were isolated and measured.

| Statistical analysis
All data shown were presented as the means ± S.D. of three independently repeated experiments. For multiple comparisons, the statistical differences were performed by one-way ANOVA or two-tailed t-test. It was regarded to be statistically significant when p-value <0.05. Graphpad Prism 6.0 was used for statistical analysis.

| Umbelliprenin induces apoptosis
One of the mainstays for antitumor therapy is apoptosis, thus we explored whether umbelliprenin could induce apoptosis. The FACS results showed that the percentage of apoptotic cells were increased from 4.87% (control group) to 27.35% (umbelliprenin 40 μg/mL) after umbelliprenin treatment in BxPC3 cells and from 3.13% (control group) to 17.25% (umbelliprenin 40 μg/mL) in PANC-1 cells, respectively ( Figure 2A). In addition, umbelliprenin could dose-dependently increase the expression level of cleaved caspase-3, caspase-8, and the proapoptotic protein Bax while reducing the expression of antiapoptotic protein Bcl-2, indicating that umbelliprenin could induce apoptosis in BxPC3 cells ( Figure 2B,C).

| Umbelliprenin induces autophagy
We further examined whether umbelliprenin could trigger autophagy in BxPC3 and PANC-1 cells by measuring the autophagic markers LC3II and p62. Immunoblotting showed that umbelliprenin dose-dependently increased LC3II expression and p62 degradation ( Figure 3A). Tf-LC3 (mRFP-GFP-tandem fluorescent LC3) was used to measure the autophagic flux in BxPC3 cells, as the green or yellow dots represented early-stage autophagosomes and the red-only dots represented the late-stage matured autolysosomes (GFP fluorescence was quenched in the acidic environment). The immunostaining results showed that both the yellow and red-only dots were significantly accumulated after umbelliprenin treatment as compared with the control group (CT) ( Figure 3B). During the initiation of autophagy, Beclin1 can interact with Vps34 to form PI3K complex. This complex then promotes Vps34 lipid kinase activity and generates phosphatidylinositol-3-phosphate (PI3P). We therefore measured whether umbelliprenin regulated the expression level of Vps34 and Beclin1. The results showed that umbelliprenin could not affect Vps34 and Beclin1 expression ( Figure 3C); however, umbelliprenin significantly enhanced the interaction between Vps34 and Beclin1 ( Figure 3D).

| Umbelliprenin-induced autophagy attenuates apoptosis
The relationship between autophagy and apoptosis seems context dependent as autophagy can either prevent or promote apoptosis. To explore the role of umbelliprenininduced autophagy in BxPC3 cells, we co-incubated umbelliprenin with 3-MA, an autophagy inhibitor. As shown in Figure 4A, the accumulation of LC3II was decreased after 3-MA added in; however, umbelliprenin plus 3-MA could significantly increase the cleavage of caspase 3 as compared with umbelliprenin or 3-MA treatment alone. Consistently, the cell growth was greatly inhibited after combination treatment ( Figure 4B). To further verify the role of umbelliprenin-induced autophagy in BxPC3 cells, we generated Atg7-negative BxPC3 cells (KO) or BxPC3 cells with empty CRISPR/Cas9 plasmid as control (WT). We found that apoptosis and cell growth inhibition induced by umbelliprenin were an augment in Atg7 KO cells but not in Atg7 WT cells, suggesting that inhibition of autophagy increased umbelliprenin-induced apoptosis in BxPC3 cells ( Figure 4C,D).

| Umbelliprenin inhibits pancreatic cancer cell stemness
To determine whether umbelliprenin regulated pancreatic cancer cell stemness, BxPC3 cells were treated with umbelliprenin and followed by ALDEFLOUR assay. It showed that the proportion of ALDH+ cells in the umbelliprenin group were greatly decreased when treated with umbelliprenin ( Figure 5A). In addition, the mammosphere formation assays showed that treatment with umbelliprenin significantly reduced the size and the number of formed mammospheres in BxPC3 cells ( Figure 5B). Moreover, NANOG, OST4, and SOX2 mRNA expression levels were all downregulated after umbelliprenin added in ( Figure 5C). Therefore, it revealed that umbelliprenin significantly suppressed cancer cell stemness in pancreatic cancer.

| Umbelliprenin inhibits Akt signal pathway in pancreatic cancer
PI3K/Akt signal pathway is one of the major signal pathways in the regulation of both apoptosis and autophagy. Our results showed that umbelliprenin decreased Akt and mTOR phosphorylation level, while the total protein levels of Akt and mTOR were not significantly changed ( Figure 6A). These results indicated that Akt/ mTOR signaling pathway was involved in umbelliprenininduced autophagy.

| Umbelliprenin regulates Notch1 signal pathway in pancreatic cancer
NOTCH, βcatenin, and Hedgehog signaling pathways were important regulators to maintain CSCs. We found that umbelliprenin treatment did not change the protein expression level of βcatenin, Shh, and SUFU ( Figure 7A), indicating that Wnt/β-catenin and Hedgehog signaling pathways are not involved in the umbelliprenin-mediated inhibition of pancreatic cancer cell stemness. However, umbelliprenin could significantly reduce the expression of Notch1 intracellular domain (NICD1) but not NICD2, NICD3, and NICD4 in BxPC3 cells ( Figure 7B). In addition, the NICD1 expression in BxPC3 mammospheres was decreased after umbelliprenin treatment ( Figure 7C). These results indicated the importance of Notch1 signaling pathway in the umbelliprenin-regulated inhibition of pancreatic cancer cell stemness.

| Umbelliprenin inhibits tumor growth in vivo
To further explore whether umbelliprenin could inhibit pancreatic tumor growth in vivo, a xenograft model was established using BxPC3 cells. The treatment schema is summarized in Figure 8A. BxPC3 cells were injected subcutaneously into the flank of each nude mouse. When the tumor volume reached about 100 mm 3 , the nude mice were assigned into five groups. We found that umbelliprenin suppressed BxPC3 tumor growth as compared with control group. Umbelliprenin in combination with 3-MA greatly reduced the tumor burden compared with the umbelliprenin group or even the gemcitabine group ( Figure 8B-D). However, the body weight of these mice had no significant difference ( Figure 8E). Moreover, we measured the expression levels of apoptosis, autophagyrelated proteins, and Notch1 pathway in these tumor samples. It revealed that umbelliprenin upregulated the expression of LC3II, the cleavage of caspase 3, downregulated the expression level of p-Akt and NICD1 ( Figure 8F). Collectively, these data suggested that umbelliprenin F I G U R E 4 Umbelliprenin-induced autophagy attenuated apoptosis. BxPC3 cells incubated with umbelliprenin (10 μg/mL) and/or 3-MA (5 mM) were used for western blot assay (A) and MTT assay (B) as indicated. Atg7-WT and Atg7-KO BxPC3 cells were treated with umbelliprenin then western blot assay (C) and MTT assay (D) were performed as indicated. *p < 0.05, **p < 0.01.

F I G U R E 5
Umbelliprenin reduced pancreatic cancer cell stemness. BxPC3 cells were treated with/without umbelliprenin (10 μg/mL). (A) ALDEFLUOR assay was carried out. (B) The cells self-renewal ability was evaluated by tumor-sphere formation assay. (C) The mRNA level of stem cell markers was analyzed by reverse transcription-quantitative PCR. *p < 0.05, **p < 0.01, and ***p < 0.001. induced cell apoptosis and autophagy inhibits cancer cell stemness in vivo. The combination of 3-MA with umbelliprenin significantly reduced tumor burden as compared with gemcitabine or umbelliprenin treatment alone.

| DISCUSSION
Coumarins and their derivatives are ubiquitous in nature and exert various anticancer mechanisms. Coumarin derivatives like irosustat are under clinical trials to treat cancer, suggesting their potential in the development of novel drugs. 26 Umbelliprenin is a sesquiterpene coumarin extracted from A. absinthium It has also been found in various common dietary sources such as celery, Coriandrum sativum, and citrus limon. 27 Previous studies demonstrated that umbelliprenin inhibits cancer cell proliferation in various cancer cells. However, its antitumor effect in pancreatic cancer has not been reported. In the current study, we found that umbelliprenin dose-and timedependently inhibited pancreatic cancer cell and tumor growth in vitro and in vivo. Apoptosis is recognized as prototypical Type I programmed cell death, which is regulated by both the intrinsic and extrinsic pathways. Caspases are cleaved and activated during the apoptotic process. The proapoptotic protein Bax and antiapoptotic protein Bcl-2 can regulate the apoptotic mitochondrial pathways by regulation the release of cytochrome c from mitochondria. 28 In this study, we found that umbelliprenin increased the cleavage of caspase3 and caspae8 and Bax; while decreasing the expression of Bcl-2. Furthermore, the percentage of Annexin V/PI-positive cells was upregulated after umbelliprenin treatment, suggesting that umbelliprenin-induced apoptosis in BxPC3 cells through both intrinsic and extrinsic pathways. Consistently, previous studies showed that umbelliprenin could induce apoptosis in various cancer cells. [19][20][21]29 Autophagy is an evolutionarily conserved catabolic process induced by various cellular stress, which inhibits cell damage and helps tumor cell survival. During this process, LC3 was conjugated with phosphatidylethanolamine (PE) to form lapidated LC3 (LC3II) and stably associated with the autophagosome membrane. Therefore, LC3II is widely used as an autophagic biomarker. Another autophagic biomarker is p62, which interacts with LC3 and transports into the autolysosome for degradation. 30 The Vps34 complex, consisting of Vps34, Beclin1, and Vps15, plays a pivotal role in the initiation of autophagosome formation. In this complex, Bcelin1 interacted with Vps34 to promote its lipid kinase activity, thereby promoting the synthesis of PI3P and increasing autophagosome formation. 31 Our findings showed that umbelliprenin could increase LC3II accumulation, promote p62 degradation in vitro and in vivo. Although umbelliprenin could not increase Vps34 or Beclin1 expression, it could significantly increase Vps34-Beclin1 interaction. These results suggested that umbelliprenin induced autophagy in BxPC3 cells.
The relationship between coumarin-induced autophagy and apoptosis are complicated. Coumarins such as compound 8b (a hybrid of coumarin and phenylsulfonylfuroxan), daphnetin, and urolithins could induce autophagy to help cell survival [32][33][34] ; while coumarins and their derivatives such as Psoralidin, 3-benzyl coumarin seco-B-ring derivative and Ferulin C induced autophagic cell death. [35][36][37] Thus, it is necessary to examine the role of umbelliprenininduced autophagy in pancreatic cancer. In this study, we showed that umbelliprenin-induced cell growth inhibition and apoptosis was augmented after blocking autophagy by 3-MA. Moreover, the number of caspase3 cleavage was much more in Atg7-KO BxPC3 cells than in Atg7-WT BxPC3 cells after umbelliprenin treated. These results suggested that umbelliprenin-induced autophagy plays a cytoprotective role in pancreatic cancer cells.
Pancreatic CSCs have been involved in tumor initiation, metastases, recurrence, and drug resistance. Stemnessassociated biomarkers like SOX2, OCT4, Nanog, and increased ALDH activity are responsible for cancer stem cell self-renew and differentiation and correlated with poor prognosis in pancreatic cancer patients. 38 In the present study, we observed that umbelliprenin greatly decreased the subpopulation of ALDH+ pancreatic CSCs and reduced the number and size of formed stem cell spheres. Umbelliprenin treatment significantly downregulated the mRNA of Oct4, Nanog, and SOX2, suggesting umbelliprenin as a putative pancreatic cancer stem cell killing drug.
Akt signal pathway plays a critical role in cell cycle, apoptosis and autophagy. 39 Many antitumor chemicals are reported to induce autophagy and apoptosis in cancer cells through Akt signal pathway. 40,41 In the present study, we showed that umbelliprenin significantly reduced the phosphorylation levels of AKT and mTOR. These results demonstrated that umbelliprenin-mediated induction of apoptosis and autophagy might be involved in Akt signaling pathway inhibition.
We also revealed that umbelliprenin exhibited the potential in decreasing pancreatic CSCs by inhibiting Notch1 signal pathway. Notch signal pathway plays a critical role in the mediation of the pancreatic CSC phenotype during cancer progression. 42 While silencing Notch1 results in the inhibition of cell proliferation, reduction of ALDH+ cell, and induction of apoptosis and gemcitabine resistance in pancreatic cancer. 43 We showed that umbelliprenin treatment significantly reduced NICD1 expression in BxPC3 cells but not NICD2, NICD3, or NICD4 expression. In addition, NICD1 protein expression was downregulated in BxPC3 mammospheres after umbelliprenin added in. These results suggested that Notch1 signaling pathway played a critical role in umbelliprenin-mediated pancreatic cancer cell stemness.

| CONCLUSION
In summary, our study showed that umbelliprenin significantly inhibited pancreatic tumor cell growth in vitro and in vivo. In addition, umbelliprenin could induce apoptosis and cyto-protective autophagy by blocking Akt signaling pathway. Umbelliprenin could diminish pancreatic CSCs by inhibiting Notch1 signaling pathway. It suggests a promising application of umbelliprenin combinate with 3-MA as an adjunctive agent for the treatment of pancreatic cancer.