Activation of AMP-Activated Protein Kinase α and Extracelluar Signal-Regulated Kinase Mediates CB-PIC-Induced Apoptosis in Hypoxic SW620 Colorectal Cancer Cells

Here, antitumor mechanism of cinnamaldehyde derivative CB-PIC was elucidated in human SW620 colon cancer cells. CB-PIC significantly exerted cytotoxicity, increased sub-G1 accumulation, and cleaved PARP with apoptotic features, while it enhanced the phosphorylation of AMPK alpha and ACC as well as activated the ERK in hypoxic SW620 cells. Furthermore, CB-PIC suppressed the expression of HIF1 alpha, Akt, and mTOR and activated the AMPK phosphorylation in hypoxic SW620 cells. Conversely, silencing of AMPKα blocked PARP cleavage and ERK activation induced by CB-PIC, while ERK inhibitor PD 98059 attenuated the phosphorylation of AMPKα in hypoxic SW620 cells, implying cross-talk between ERK and AMPKα. Furthermore, cotreatment of CB-PIC and metformin enhanced the inhibition of HIF1α and Akt/mTOR and the activation of AMPKα and pACC in hypoxic SW620 cells. In addition, CB-PIC suppressed the growth of SW620 cells inoculated in BALB/c athymic nude mice, and immunohistochemistry revealed that CB-PIC treatment attenuated the expression of Ki-67, CD34, and CAIX and increased the expression of pAMPKα in CB-PIC-treated group. Interestingly, CP-PIC showed better antitumor activity in SW620 colon cancer cells under hypoxia than under normoxia, since it may be applied to chemoresistance. Overall, our findings suggest that activation of AMPKα and ERK mediates CB-PIC-induced apoptosis in hypoxic SW620 colon cancer cells.


Introduction
Hypoxia is a critical factor of solid tumors that renders tumor cells resistant to some chemotherapeutic agents [1]. Most of the cancer cells grow fast and suffer nutrition and oxygen deficiency under hypoxia [2]. Under hypoxic condition, the cells are regulated by HIF-1 and other related signaling including AMP-activated protein kinase (AMPK) pathway. AMPK consists of catalytic subunit and regulatory and subunits, which act for cellular adaptation to ATP-consuming stimuli such as glucose deprivation [3]. When cells need to overcome metabolic stress, the ratio of AMP to ATP is markedly increased by AMPK [4]. Thus, activation of AMPK suppresses metabolic functions that consume or generate ATP [5,6], and decreased activation of AMPK significantly increases the risk of cancer in several animal models [7][8][9]. Also, the activation of AMPK suppresses survival signals such as phosphoinositide-3-kinase (PI3K)/AKT signaling and regulates mTOR pathway [10]. Recently, metformin as an old antidiabetic drug emerges as anticancer agent via AMPK inhibition [11].

Cell
Culture under Hypoxia. SW620 cells were seeded in 100 mm Falcon plates at 1 × 10 6 cells/plate in RPMI 1640 supplemented with 10% FBS and 1% penicillin/streptomycin. The cells were cultured at 37 ∘ C in a humidified atmosphere containing from 5% CO 2 to 60-80% confluence and then used for Western blot analysis. For the hypoxic treatment, the cells were transferred under an anaerobic chamber (Forma Scientific, OH, USA) with a humidified atmosphere of 2% O 2 and 5% CO 2 balanced with N 2 and exposed to various concentrations of CB-PIC for 2∼24 hours.

AMPK Transfection
Assay. SW620 cells were transfected with siRNA control or AMPK by using Polyplus siRNA transfection reagent (Illkirch, France) according to manufacturer instructions and then treated with CB-PIC for 4 hr under hypoxia. In brief, siRNA (100 pmol) was mixed with transfection reagent in Opti-MEM serum-free media (Invitrogen) and incubated for 15 min at room temperature. The siRNA/transfection reagent mixture was added to the cells for 48 hr. Medium was changed before CB-PIC treatment under hypoxia.

Animals and CB-PIC Treatment.
Six-week-old male BALB/c athymic nude mice (25 ± 3 g) were purchased from Chung-Ang Laboratory Animals (Seoul, Korea) and housed in animal facility at 22 ± 3 ∘ C and 60 ± 10% humidity with light-controlled (12 h, 07:00-19:00) environment. All materials including bedding and feed were sterilely cleaned by UV rays for 15 min before treated to the mice. The experiments were conducted in accordance with the guidelines approved by Institutional Animal Care and Use Committee, Kyung Hee University (KHUASP(SE)-11-005). CB-PIC was diluted in 4% Tween 20 normal saline. After 1 week adaptation, the animals were assigned to four groups ( = 6): normal control (vehicle), negative control (vehicle (saline) + SW620 inoculation), CB-PIC20 (20 mg/kg + SW620 inoculation), and CB-PIC50 (50 mg/kg + SW620 inoculation). CB-PIC solved in saline and Tween 20 was daily injected into the abdomen of mice for 20 days.

SW 620 Xenograft
Model. The animal study was conducted under guidelines approved by Institutional Animal Care and use Committee, Kyung Hee University [KHUASP(SE)-11-005] as previously described with minor modifications [18]. Briefly, 2 × 10 6 of SW 620 cells were mixed with Matrigel (Becton Dickinson, 50% in 100 L) and injected subcutaneously into the right flank of 6-week-old male BALB/c athymic nude mice (Chung-Ang Laboratory Animals, Seoul, Korea)) for 3 groups (Control and CB-PIC groups). Three day after SW620 cell inoculation, 2% Tween-80/saline was daily injected into the mice of control group, while CB-PIC (20 and 50 mg/kg) dissolved in 2% Tween-20 was injected into the mice of CB-PIC groups. Tumor size was monitored twice a week with a caliper, and tumor volume was also calculated as described [18]. At the end of animal study, tumors were dissected, weighed, and photographed. A piece of each tumor was fixed in 10% phosphate-buffered formalin (PBS) for immunohistochemistry.
2.11. Immunohistochemistry. For immunohistochemistry, paraffin sections (4 m) from tumors dissected were stained with hematoxylin and eosin. Immunohistochemical staining was performed in tumor sections using the indirect avidin biotin-enhanced horseradish-peroxidase method. Antigen retrieval was performed after dewaxing and dehydration of the tissue sections by microwave for 10 min in 10 mM citrate buffer. Tumor sections were cooled at room temperature, treated with 3% hydrogen peroxide in methanol for 10 min, and blocked with 6% horse serum for 30 min at room temperature in humidity chamber. Sections were then incubated with the primary antibody against to Ki-67 ( For semiquantitation, ten photomicrographs (200×) were taken with a CCD camera, avoiding gross necrotic areas. The positively stained cells/vessels within each photomicrograph were counted. The counting of total cancer cells was aided with the ImagePro+ image processing program.

Data Analyses.
Every experiment was repeated at least three times. Data were shown as means ± SE. Significant differences were evaluated using Student's -test and a Tukey-Kramer multiple comparison posttest.

CB-PIC Exerts Significant Cytotoxicity in Hypoxic SW620
Cancer Cells. The hypoxic cells are considered more aggressive and resistant to various therapies including radiation or chemotherapy [19,20]. Therefore, the cytotoxic effects of CB-PIC were evaluated in human SW 620 colorectal cancer cells under hypoxia or normoxia. CB-PIC showed significant cytotoxicity in SW 620 cells better than in HT29 and HCT116 cells (Figure 1(b)). Interestingly, the proliferation of SW 620 cells was increased under hypoxia compared to normoxia. However, CB-PIC dramatically suppressed the viability of SW620 cells even under hypoxia (Figure 1(c)).

CB-PIC Induces Apoptosis in Hypoxic SW620 Cancer
Cells. To find out whether the cytotoxicity was due to apoptosis or necrosis, cell analysis was performed. CB-PIC significantly increased sub-G1 population in SW620 cells in a time-dependent manner in normoxia or hypoxia (Figures  2(a) and 2(b)). Consistently, CB-PIC significantly increased TUNEL positive cells (Figure 3(c)), cleaved PRAP, and attenuated the expression of Bcl-2 (Figure 3(a)) in SW620 cells under normoxia or hypoxia by Western blotting. In addition, the activity of the caspase-3 was increased by treatment of CB-PIC for 6 h in SW 620 cells (Figure 3(b)).

CB-PIC Activated the Phosphorylation of AMPK and ERK in Hypoxic SW620 Cancer Cells.
It is well known that the decreased AMPK activation is implicated in metabolic disorders with high cancer risk [21]. CB-PIC at 40 g/mL activated the phosphorylation of AMPK and ERK in hypoxic SW620 cells (Figures 3(a) and 4(a)) and enhanced HIF1 accumulation up to 4 h in hypoxic SW620 cancer cells. In contrast, CB-PIC activated the phosphorylation of AMPK and ERK both normoxia and hypoxia. Regarding the effect of ERK activation of cell cycle analysis and apoptosis, Tang and his colleagues suggested that low intensity DNA damageinduced ERK activation causes cell cycle arrest, while extensive DNA damage-induced ERK activation causes apoptosis [22]. CB-PIC induced PARP cleavage and ERK phosphorylation in SW620 cancer cells even after 6 h culture (Figure 3(a)). Similarly, CB-PIC at 40 g/mL activated the phosphorylation of AMPK and ERK in a time-dependent manner under hypoxia (Figure 4(a)) rather than under normoxia. However, HIF1 and pAMPK tended to be downregulated after 4 h culture, while the phosphorylation of ERK was consistently increased by CB-PIC in hypoxic SW620 cells (Figure 4(a)). In contrast, pJNK and pp38 were not altered by CB-PIC under hypoxia and normoxia (data not shown).

ERK Inhibitor PD98059 Attenuated Phosphorylation of AMPK and Silencing of AMPK Blocked Phosphorylation of ERK in Hypoxic SW620 Cancer Cells.
To elucidate the underlying mechanism between ERK and AMPK , ERK inhibitor PD98059 and AMPK siRNA transfection were used in hypoxic SW620 cells. ERK inhibitor PD98059 attenuated phosphorylation of AMPK and ERK, while silencing of AMPK blocked phosphorylation of ERK and AMPK by CB-PIC in hypoxic SW620 cancer cells (Figures 5(a) and 5(b)).

CB-PIC Enhanced Phosphorylation of AMPK and ERK
Induced by Metformin in Hypoxic SW620 Cancer Cells. As metformin is known as AMPK activator [23,24] in SW620 cells under hypoxia, treatment with 5 mM metformin for 2 hours increased the phosphorylation of AMPK (Figure 6(a)). In addition, cotreatment of CB-PIC and metformin enhanced phosphorylation of ACC, AMPK , and pERK and also suppressed phosphorylation of mTOR and Akt and accumulation of HIF-1 in hypoxic SW620 cells (Figure 6(b)).

CB-PIC Inhibited the Growth of SW620 Cancer Cells Inoculated in BALB/c Athymic Nude
Mice. CB-PIC suppressed the growth of SW620 cancer cells inoculated in BALB/c athymic nude mice at doses of 20 mg/kg and 50 mg/kg without affecting body weight (Figures 7(a), 7(b), and 7(c)). Consistently, immunohistochemistry revealed that CB-PIC treatment attenuated the expression of expression of Ki-67(proliferation), CD34 (blood vessel density), and carbonic anhydrase(CA)IX (hypoxic marker) and increased pAMPK index in the tumor sections of CB-PIC-treated group (50 mg/kg) compared to untreated control (Figure 7(d)).

Discussion
Since clinically nearly 50∼60% of solid tumors have more hypoxic regions compared to normal cells [25] and hypoxic cells are more aggressive and resistant to various cancer treatment [26,27], it is crucial to regulate hypoxia signals.
Evidence-Based Complementary and Alternative Medicine  Thus, in the current study, molecular mechanism of CB-PIC was investigated in SW620 colon cancer cells under hypoxia. We found CB-PIC exerted significant cytotoxicity in SW620 cells better than HCT116 and HT29 colon cancer cells under hypoxia. We found the cytotoxicity of CB-PIC was caused by apoptosis, not necrosis, through the significant increase of sub-G1 accumulation, TUNEL positive cells, and PARP cleavage in hypoxic SW620 cells. AMP-activated protein kinase (AMPK), a cellular energy sensor conserved in eukaryotes, inhibits energy-consuming processes and activates energy-producing processes to restore the energy homeostasis inside the cell. Thus, AMPK activators, such as metformin and thiazolidinediones used for the treatment of type II diabetes, inhibit tumorigenesis through regulation of cell growth, cell proliferation, autophagy, stress responses, and cell polarity [5,28]. Likewise CB-PIC enhanced the phosphorylation of AMP-activated protein kinase (AMPK) alpha and its substrate acetyl-CoA carboxylase (ACC) hypoxic SW620 colon cancer cells. ERK1/2 as one of MAPK proteins can be activated transiently or persistently by MEK1/2 and upstream MAP3Ks. In general, activation of ERK1/2 generally promotes cell survival, but under certain conditions, ERK1/2 can have proapoptotic functions [22,29]. Also, some evidence suggests that ERK has function of proapoptotic characteristics under certain conditions, and other animal study supports that ERK can induce apoptosis [29,30]. Activation of ERK can suppress the expression of phosphatidylinositol-3-kinase/ Akt survival pathway [31]. ERK and Akt reported to share some multimolecular complexes at least ERK1/2, Akt, ribosomal S6 kinase 1, and phosphoinositide-dependent kinase 1 [32]. In the present study, CB-PIC also activated ERK to induce apoptosis in hypoxic SW620 cells.
Furthermore, CB-PIC suppressed the expression of hypoxia inducible factor 1 (HIF1) alpha as a hypoxia master regulator [33], Akt, and mammalian target of rapamycin (mTOR) similar to AMPK activator metformin in hypoxic SW620 cells, implying the important roles of mammalian target of rapamycin (mTOR) and AMPK signaling in cancer metabolism [34,35].
To find out the critical roles of AMPK and ERK, ERK inhibitor PD 98059 and AMPK siRNA were used to evaluate their effects on CB-PIC-induced signaling in hypoxic SW620 cells. Silencing of AMPK blocked PARP cleavage, and ERK activation induced by CB-PIC, while ERK inhibitor PD 98059 attenuated the phosphorylation of AMPK in hypoxic SW620 cells, suggesting the possibility of cross-talk between ERK and AMPK . Furthermore, cotreatment of CB-PIC and metformin enhanced inhibition of HIF1 and Akt/mTOR and activation of AMPK and pACC in hypoxic SW620 cells.
In addition, CB-PIC at 20 and 50 mg/kg suppressed the growth of SW620 cancer cells implanted in BALB/c athymic nude mice. Consistently, immunohistochemistry (IHC) showed that CB-PIC treatment attenuated the expression of decreased expression of Ki-67(proliferation), CD34 (blood vessel density), and carbonic anhydrase(CA)IX (hypoxic marker) and increased expression of pAMPK index in CB-PIC-treated group compared to untreated control, indicating that CB-PIC exerts antitumor activity via inhibition of proliferation, angiogenesis, and hypoxia along with AMPK activation and apoptosis induction in SW620 cancer cells.
Given that HIF1 , a major target molecule in hypoxia is closely associated with multidrug resistance [36,37], it is more significant that CB-PIC exerts antitumor activity in hypoxic SW620 cancer cells, since it can be applicable to cancer cells under hypoxic microenvironment with high risk of chemoresistance. Thus, it is also necessary for us to perform further study on the inhibitory effect of CB-PIC on MDR related proteins in resistant cancer cells in vitro and in vivo in the future.
In summary, CB-PIC showed significant cytotoxicity against SW620 colon cancer cells and induced apoptosis by sub-G1 accumulation, the cleavage of PARP and caspase 3 and increased TUNEL positive cells in SW620 cancer cells. Interestingly, CB-PIC increased phosphorylation of ERK, AMPK , and ACC, attenuated the expression of HIF1 and Akt/mTOR and also enhanced the antitumor activity of metformin by potentiating inhibition of HIF1 and Akt/mTOR and activation of AMPK and pACC. Also, CB-PIC suppressed in vivo growth of SW620 cancer cells, and IHC showed decreased expression of Ki-67, CD34, and CAIX and increased expression of pAMPK . Overall, our findings suggest that activation of AMPK and ERK mediates CB-PIC-induced apoptosis in hypoxic SW620 colon cancer cells.