Sorafenib in Combination with Betulinic Acid Synergistically Induces Cell Cycle Arrest and Inhibits Clonogenic Activity in Pancreatic Ductal Adenocarcinoma Cells

Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers in the world due to late diagnosis and poor response to available treatments. It is important to identify treatment strategies that will increase the efficacy and reduce the toxicity of the currently used therapeutics. In this study, the PDAC cell lines AsPC-1, BxPC-3, and Capan-1 were treated with sorafenib and betulinic acid alone and in combination. We examined the effect of combined treatments on viability (MTS test), proliferation and apoptosis (annexin V staining), cell cycle arrest (PI staining), alterations in signaling pathways (Western blotting), and colony-forming ability. The combination of sorafenib with betulinic acid inhibited the viability and proliferation of PDAC cells without the induction of apoptosis. The antiproliferative effect, caused by G2 cell cycle arrest, was strongly associated with increased expression of p21 and decreased expression of c-Myc and cyclin D1, and was induced only by combined treatment. Additionally, decreased proliferation could also be associated with the inhibition of the P13K/Akt and MAPK signaling pathways. Importantly, combination treatment reduced the colony-forming ability of PDAC cells, as compared to both compounds alone. Collectively, we showed that combined treatment with low concentrations of sorafenib and betulinic acid had the capacity to inhibit proliferation and abolish clonogenic activity in PDAC cell lines.


Introduction
Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers of the digestive system worldwide [1]. Pancreatic cancer affects both men and women, and the overall five-year survival rate remains below 5% [2]. Chemotherapy is an important therapeutic method, but the sensitivity is low due to increasing drug resistance [3]. This situation is probably caused by the specific tumor microenvironment of pancreatic cancer.
Currently, gemcitabine is used as a standard therapy for advanced pancreatic cancer. However, gemcitabine alone is not very effective and is associated with drug resistance [4]. In view of that problem, developing new agents and innovative approaches are a continuing research effort to advance the treatment of this disease [5].
We have previously shown that combination treatment with sorafenib (SOR) and betulinic acid (BA) inhibits proliferation, induces cell death, and reduces colony-forming ability in non-small cell lung cancer (NSCLC) cell lines with different KRAS mutations [6]. In pancreatic cancer, activating KRAS mutations occur at a frequency of 90% [7]. Previous studies have shown that almost all therapies targeting KRAS mutations have failed [8]. Many efforts have been made in the field of PDAC therapies to develop drugs or combination of drugs targeting the components of the downstream effector pathways of KRAS signals, such as the MAPK and PI3K/Akt signaling pathways [9]. Sorafenib (SOR) is an oral, multitargeted kinase inhibitor directed against the mitogen-activated protein kinase (MAPK) pathway, vascular endothelial growth factor receptor-2 (VEGFR-2) and -3 (VEGFR-3), platelet-derived growth factor receptor-b (PDGFRb), Fms-related tyrosine kinase 3 (FLT3), and mast/stem cell growth factor receptor Kit (c-KIT) [10,11]. Sorafenib is approved for the treatment of renal cell carcinoma, hepatocellular carcinoma, and thyroid cancer [12,13]. The data suggest that sorafenib has a potential therapeutic benefit for PDAC treatment; however, the results of a combination therapy utilizing sorafenib with gemcitabine or sorafenib with erlotinib indicated that sorafenib was not able to enhance chemotherapeutic effect [14,15].
Several anticancer and anti-infectious drugs are derived from natural products [16]. Betulinic acid (BA) is a natural pentacyclic triterpene with a lupine structure isolated from the bark of the white birch (Betula pubescens) [17]. BA has been shown to induce apoptosis in a p53-and caspase-independent manner, mitochondrial membrane alteration, and DNA fragmentation [18]. Importantly, some reports showed that BA induced cell death by downregulating the expression of the P13K/Akt signaling pathway [19]. BA exhibits significant antitumor activities in various cancer cells, including pancreatic cancer [20].
In this study, we showed that combination treatment with SOR and BA also reduced the clonogenic potential of PDAC cells, suppressed proliferation via cell cycle arrest, and inhibited the PI3K/Akt and MAPK signaling pathways, but it did not induce apoptosis. Combination therapies that act on different molecular targets in the cancer should increase the probability of cancer elimination and decrease the development of resistant cancer cells.
Additionally, we used the annexin V-FIC/PI double staining and apoptosis-associated DNA fragmentation by staining cells with propidium iodide (PI) to evaluate whether the SOR and BA combination induced apoptosis in PDAC cells. As shown in Figure 2, combination treatment did not increase apoptosis in PDAC cell lines.

The Combination of Sorafenib and Betulinic Acid Induces G2 Cell Cycle Arrest in AsPC-1 Cells
The cell cycle distribution analysis was performed using flow cytometry to elucidate how the combination of SOR and BA inhibited cell proliferation. The results showed that the combination of SOR and BA significantly induced cell cycle arrest at G2 phase ( Figure 3A). The percentage of G2 phase cells increased to 39% after treatment with the SOR and BA combination. Capan-1 cells after treatments with sorafenib (AsPC-1 and Capan-1: 5 µM, BxPC-3: 3 µM) and betulinic acid (6 µM) alone and in combination (n = 3). Data are presented as means ± SD. * p < 0.05 compared with the sorafenib treatment group and betulinic acid treatment group.

The Combination of Sorafenib and Betulinic Acid Induces G2 Cell Cycle Arrest in AsPC-1 Cells
The cell cycle distribution analysis was performed using flow cytometry to elucidate how the combination of SOR and BA inhibited cell proliferation. The results showed that the combination of SOR and BA significantly induced cell cycle arrest at G2 phase ( Figure 3A). The percentage of G2 phase cells increased to 39% after treatment with the SOR and BA combination.
The effect was further confirmed by the detection of key proteins that help regulate the cell cycle. Figure 3B shows that the level of p21 increased after treatment with SOR and BA alone and in combination for 24 h, while the levels of c-Myc and cyclin D1 decreased after combination treatment. However, the expression of cyclin B1 remained unchanged. These results suggest that cell cycle arrest in the G2 phase is a probable mechanism by which SOR + BA prevent PDAC cell proliferation. The results were similar in the other two cell lines. The effect was further confirmed by the detection of key proteins that help regulate the cell cycle. Figure 3B shows that the level of p21 increased after treatment with SOR and BA alone and in combination for 24 h, while the levels of c-Myc and cyclin D1 decreased after combination treatment. However, the expression of cyclin B1 remained unchanged. These results suggest that cell cycle arrest in the G2 phase is a probable mechanism by which SOR + BA prevent PDAC cell proliferation. The results were similar in the other two cell lines.

Combination Treatment with Sorafenib and Betulinic Acid Inhibits the Expression of the PI3K/Akt and MAPK Signaling Pathways in the AsPC-1 and BxPC-3 Cell Lines
We investigated the effects of SOR and BA alone and in combination on the PI3K/Akt and/or MAPK signaling pathways in AsPC-1 and BxPC-3 cells, because the activation of these pathways is important for cell cycle progression in human pancreatic cancer cells [23,24]. Western blotting results showed ( Figure 4) that combination treatment inhibited ERK1/2 phosphorylation after 24 and 72 h in BxPC-3 cells. In addition, combination treatment inhibited the expression and phosphorylation of Akt after 72 h in AsPC-1 cells and after 24 and 72 h in BxPC-3 cells. . Actin served as a loading control. Data are presented as means ± SD. * p < 0.05, ** p < 0.01 compared with the sorafenib treatment group and betulinic acid treatment group. All experiments were repeated at least three times.

Combination Treatment with Sorafenib and Betulinic Acid Inhibits the Expression of the PI3K/Akt and MAPK Signaling Pathways in the AsPC-1 and BxPC-3 Cell Lines
We investigated the effects of SOR and BA alone and in combination on the PI3K/Akt and/or MAPK signaling pathways in AsPC-1 and BxPC-3 cells, because the activation of these pathways is important for cell cycle progression in human pancreatic cancer cells [23,24]. Western blotting results showed ( Figure 4) that combination treatment inhibited ERK1/2 phosphorylation after 24 and 72 h in BxPC-3 cells. In addition, combination treatment inhibited the expression and phosphorylation of Akt after 72 h in AsPC-1 cells and after 24 and 72 h in BxPC-3 cells.

The Combination of Sorafenib and Betulinic Acid Reduces the Colony-Forming Ability of PDAC Cell Lines
The long-term assay (clonogenic survival) was employed to determine the ability of combination treatment with SOR and BA to influence pancreatic cancer cell survival. PDAC cell lines were treated

The Combination of Sorafenib and Betulinic Acid Reduces the Colony-Forming Ability of PDAC Cell Lines
The long-term assay (clonogenic survival) was employed to determine the ability of combination treatment with SOR and BA to influence pancreatic cancer cell survival. PDAC cell lines were treated with sorafenib (AsPC-1 and Capan-1: 5 µM, BxPC-3: 3 µM) and betulinic acid (6 µM) alone or in combination for 14 days, after which the number of colonies formed was counted ( Figure 5). The SOR and BA combination significantly reduced the number of colonies (surviving fraction) compared to the control and single sorafenib and betulinic acid treatments for all cell lines. In addition to reducing the number of colonies, colony size appeared to be smaller in the SOR+BA treatment, suggesting that the combination also prevented the clonogenic expansion of existing tumor cells.

Discussion
There are limited effective treatments for patients with pancreatic cancer. Gemcitabine combined with other adjuvants has been used to treat PDAC, but these approaches have had limited success and only extended life span by months with additional toxicity [25,26]. Several combination therapies with sorafenib, such as SOR with gemcitabine and erlotinib, have been evaluated in clinics; however, the outcomes were disappointing [14,15,27,28]. In addition, SOR has already been shown to exert no benefit for survival or other efficacy parameters in locally advanced or metastatic pancreatic adenocarcinoma [29]. Furthermore, Pandita et al. [20] showed that the combination of betulinic acid and gemcitabine inhibited cell proliferation, induced apoptosis, and downregulated the expression of PKM2 in PDAC cell lines. Moreover, betulinic acid was shown to have broad antitumor effects on PDAC cells in some other studies [30][31][32].
In this study, we determined that the natural product betulinic acid enhanced the effects of sorafenib in PDAC cell lines. Single treatment with SOR or BA shows dose-dependent growth inhibition only in the BxPC-3 cell line, whereas AsPC-1 and Capan-1 are highly resistant. Increased sensitivity of BxPC-3 cells can be caused by the presence of a mutation in BRAF, which is a possible target of sorafenib. We found that SOR in combination with BA significantly inhibited the proliferation, promoted cell cycle arrest, and reduced the colony-forming ability of PDAC cells in vitro. Interestingly, this combination did not induce apoptosis.

Discussion
There are limited effective treatments for patients with pancreatic cancer. Gemcitabine combined with other adjuvants has been used to treat PDAC, but these approaches have had limited success and only extended life span by months with additional toxicity [25,26]. Several combination therapies with sorafenib, such as SOR with gemcitabine and erlotinib, have been evaluated in clinics; however, the outcomes were disappointing [14,15,27,28]. In addition, SOR has already been shown to exert no benefit for survival or other efficacy parameters in locally advanced or metastatic pancreatic adenocarcinoma [29]. Furthermore, Pandita et al. [20] showed that the combination of betulinic acid and gemcitabine inhibited cell proliferation, induced apoptosis, and downregulated the expression of PKM2 in PDAC cell lines. Moreover, betulinic acid was shown to have broad antitumor effects on PDAC cells in some other studies [30][31][32].
In this study, we determined that the natural product betulinic acid enhanced the effects of sorafenib in PDAC cell lines. Single treatment with SOR or BA shows dose-dependent growth inhibition only in the BxPC-3 cell line, whereas AsPC-1 and Capan-1 are highly resistant. Increased sensitivity of BxPC-3 cells can be caused by the presence of a mutation in BRAF, which is a possible target of sorafenib. We found that SOR in combination with BA significantly inhibited the proliferation, promoted cell cycle arrest, and reduced the colony-forming ability of PDAC cells in vitro. Interestingly, this combination did not induce apoptosis.
Our data showed that combination treatment with SOR and BA increased the expression of p21 and simultaneously decreased the expression of c-Myc and cyclin D1. Some authors also reported that cyclin D1 was involved in G2 arrest, especially under high levels of oxidative stress [33]. Hitomi et al. suggested that the elevated cyclin D1 level in the G2 phase was a critical checkpoint for the progression of the cell cycle [34]. Suppression of cyclin D1 levels during the G2 phase promoted the inhibition of proliferation [35]. The c-MYC proto-oncogene is activated in many PDAC cases and plays a central role in many cellular processes, such as proliferation, differentiation, and apoptosis [36]. Moreover, c-Myc overexpression was reported to be associated with gemcitabine resistance [37]. According to some reports, c-Myc inhibition was not always associated with cell death [38]; in vivo, c-Myc correlated inversely with p21 expression. The level of c-Myc determines if p21 is induced or suppressed and whether cells undergo apoptosis or growth arrest [39]. Increased p21 expression is associated with cell cycle arrest, proliferation inhibition, and cell senescence [40]. Cell cycle regulators, including p21, p27, and cyclins, should be tightly controlled; p21 coordinates with p27, thereby modulating the expression of cyclin D1 and E2 [40]. Deregulation of cyclin D1 can lead to genetic instability in in vitro and in vivo tumorigenesis [41]. Additionally, cyclin D1 overexpression is an independent prognostic factor for survival in patients with PDAC [42].
PI3K/Akt and MAPK/ERK are two of the early signaling pathways of cell cycle progression [28,43]. In this study, we demonstrated that combination treatment with SOR and BA inhibited pancreatic cancer cell cycle progression by inactivating PI3K/Akt and MAPK signaling. Recently published data have shown a synergic effect between sorafenib and HS-173 (a novel PI3K inhibitor). This treatment, by synergistically inhibiting the MAPK and PI3K/Akt pathways, induced G2/M arrest and increased apoptosis in pancreatic cancer cells [44]. However, while concurrent treatment with MEK and PI3K inhibitors has recently been investigated in mouse models of pancreatic cancer, only low antitumor activity was observed [45,46]. Moreover, a clinical trial of MEK and PI3K inhibitor combinations also suggested that normal tissue toxicity may limit this combination [40]. A clinical study of Akt and ERK inhibitor combinations needs to be conducted to determine whether targeting ERK rather than MEK will overcome these limitations.
Mechanisms of growth suppression by signaling inhibitors are usually characterized using short-term analyses, yet clinical application of such inhibitors involves persistent long-term treatment. In the present study, we performed a long-term clonogenic survival assay. Combination treatment with SOR and BA markedly decreased the colony-forming capability of PDAC cells. This treatment has been shown previously to be nontoxic for normal human peripheral blood lymphocytes [6]. It should be noted that tumor cell cloning efficiency is positively correlated with proliferation and self-renewal abilities, which may be associated with cell tumorigenesis [47]. Studying tumor clonogenic/stem-like cells contributes to the identification of molecular targets important in successful cancer therapy [48].
In summary, we have demonstrated for the first time that combination treatment with SOR and BA can more effectively attenuate cell proliferation, promote cell cycle arrest, and reduce colony-forming ability than a single treatment in human PDAC cell lines. Moreover, combined treatment with SOR and BA can inhibit phosphorylation of Akt and ERK1/2 more potently than their individual use, which may account for the synergistic antitumor effect of this combination treatment. This study may provide a novel indication for pancreatic cancer treatment.

Cell Viability Assay
Cell viability was assessed by the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA), according to the manufacturer's protocol. Each treatment within a single experiment was performed in triplicate. Absorbance at 490 nm was recorded using a Wallac 1420 VICTOR2 plate reader (PerkinElmer, Waltham, MA, USA). Data were normalized to the untreated control.

Cell Count
Floating and trypsinized cells were collected and suspended in fresh medium at room temperature. Twenty microliters of cell suspension was mixed with 20 µL of 0.4% Trypan blue solution (Bio-Rad, Carlsbad, CA, USA), and 20 µL of this mixture was used to count blue (dead) and white (alive) cells.

Annexin V Staining
Apoptosis was assessed by the Annexin V Apoptosis Detection Kit (Santa Cruz Biotechnology, Dallas, TX, USA), according to the manufacturer's protocol. Briefly, the cells were stained with annexin V-FITC (8 µg/mL) and PI (5 µg/mL) for 15 min at RT in the dark. The cells were washed with cold PBS (with Ca 2+ and Mg 2+ ) containing 2.5% FBS between the steps. Data were acquired using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed using Flowing Software 2.5.1 (Perttu Terho, Turku, Finland). Apoptosis was quantified as a percentage of both annexin V-positive and annexin V/PI-double-positive cells.

Cell Cycle and DNA Fragmentation Assay
The cells were fixed in 75% ethanol at 4 • C for 30 min, and then incubated with 50 ng/mL PI staining solution and 0.2 mg/mL RNase in the dark overnight at 4 • C. Data were acquired using a and betulinic acid treatment group vs. combinatorial treatment group, were analyzed by a two-tailed Student's t-test. The significance was assumed at * p < 0.05, ** p < 0.01.