Tumor Biol. (2014) 35:2461–2471 DOI 10.1007/s13277-013-1326-6
RESEARCH ARTICLE
Bufalin exerts antitumor effects by inducing cell cycle arrest and triggering apoptosis in pancreatic cancer cells Meiying Li & Xuejun Yu & Hui Guo & Limei Sun & Aijun Wang & Qiji Liu & Xiuwen Wang & Jisheng Li
Received: 6 August 2013 / Accepted: 14 October 2013 / Published online: 12 November 2013 # International Society of Oncology and BioMarkers (ISOBM) 2013
Abstract As one of the most aggressive human malignancies, pancreatic cancer is a leading cause of cancer-related deaths worldwide and only about 4 % of patients will live 5 years after diagnosis. Eighty to approximately eighty-five percent of patients are diagnosed with an unresectable or metastatic disease, which is correlated with poor prognosis and low survival rate. Therefore, it is tremendously significant to exploit novel chemicals to prevent and treat pancreatic cancer. Previous research and clinical studies have demonstrated that many natural products derived from traditional Chinese medicine (TCM) such as camptothecin derivatives and vinca alkaloids could be effective antitumor compounds, hinting that TCM is a promising source for developing new antitumor drugs. In this report, we investigated the effects of bufalin, a primary active ingredient of the traditional Chinese medicine Chan-Su, on pancreatic cancer cell lines PANC-1 and CFPAC1 and studied the underlying molecular mechanism. We found that exposure to bufalin could suppress the proliferation of pancreatic cancer cells time and dose dependently. We used flow cytometry to study the effects of bufalin on apoptosis and cell cycle distribution in PANC-1 and CFPAC-1 cells. The results indicated that bufalin could significantly induce both
apoptosis and G2/M cell cycle arrest in pancreatic cancer cells. With western blotting, we found that the expression level of an antiapoptotic protein heat shock protein 27 (Hsp27) and its partner molecule p-Akt was decreased upon the treatment with bufalin. Besides, bufalin activated pro-caspase-3 and procaspase-9 and modulated the expression level of Bcl-2 and Bax. These data suggested that bufalin may trigger apoptosis by targeting Hsp27, which could inhibit apoptosis by interfering with key apoptotic proteins. The influence on the level of cylinB1, CDK1, and p21 was also observed after bufalin treatment, and the relationship between Hsp27 and the cell cycle-related proteins mentioned above deserves much more research. In addition, our data showed that bufalin could enhance the growth inhibition effect of gemcitabine in above pancreatic cancer cells. Taken together, bufalin might be worthy of further study for its potential as a therapeutic agent for pancreatic cancer treatment. Keywords Pancreatic cancer . Bufalin . Antitumor . Cell cycle arrest . Apoptosis . Hsp27
Introduction Meiying Li and Xuejun Yu contributed equally to this work. M. Li : X. Yu : L. Sun : A. Wang : X. Wang (*) : J. Li (*) Department of Medical Oncology, Cancer Center, Qilu Hospital, Shandong University, Jinan 250012, China e-mail: [email protected] e-mail: [email protected] H. Guo Department of Ophthalmology, Qilu Hospital of Shandong University, Jinan 250012, China Q. Liu Department of Medical Genetics, Key Laboratory for Experimental Teratology of the Ministry of Education, Shandong University School of Medicine, Jinan 250012, China
Pancreatic cancer, one of the most aggressive and fatal human malignancies, is currently the fourth leading cause of cancer death in the USA and leads to about 227,000 deaths per year worldwide [1]. In China, pancreatic cancer is the sixth leading cause of cancer-related deaths, and the 5-year survival rate is only 1 to 3 % [2]. Despite advancements of the diagnosis and management of pancreatic cancer, the incidence and mortality of this malignancy have kept increasing in recent years. It is estimated that about 45,220 new cases of pancreatic cancer will be diagnosed in the USA in 2013, and 38,460 patients will die of this disease [3]. The poor prognosis of pancreatic cancer is mainly attributed to its hallmarks including delayed clinical
2462
presentation, tendency of early local and distant dissemination, and resistance to conventional chemo-radiation treatment strategies [4]. At present, surgical resection offers the only chance of cure for those diagnosed with cancer localized to the pancreas, and adjuvant chemotherapy after surgery is recommended to delay relapse and improve survival rate. Unfortunately, up to 85 % of patients present with advanced unresectable disease and the available effective regimens are quite limited [1]. For patients with advanced and metastatic pancreatic cancer, chemotherapy is the primary treatment modality and gemcitabine is routinely used for alleviating patients' symptoms since 1997 [5]. Besides monotherapy, gemcitabine has been investigated in combination with synergistic agents such as cisplatin, capecitabine, and erlotinib [6–9]. In addition, the regimen of FOLFIRINOX (5fluorouracil, oxaliplatin, irinotecan, and leucovorin) has shown superiority over gemcitabine monotherapy in prolonging patients' overall survival, but it is only limited for patients with good performance status [10]. Recently, the results of MPACT study showed that the median overall survival of patients who received nab-paclitaxel plus gemcitabine was 8.5 months, compared with 6.7 months for those who received gemcitabine monotherapy [11]. This is a real breakthrough in pancreatic cancer treatment. Despite above advancements in the chemotherapy of pancreatic cancer, gemcitabine still remains as the standard regimen for treating pancreatic cancer. However, only part of patients are responsive to gemcitabine treatment because of the inherent and acquired drug resistance and the adverse events could be intolerable for some of them [5, 12]. Therefore, it is urgent to identify novel effective anticancer drug to treat this deadly malignancy. Poisonous Chinese herbal medicine (PCHM) which is derived from botanicals, minerals, and animals has been historically used in cancer therapies by skilled Chinese practitioners for thousands of years. Numerous researches mining novel anticancer drugs from PCHM-derived natural products proved that PCHM is a huge and noteworthy reservoir for novel efficacious anticancer agents [13]. ChanSu, one such Chinese herbal medicine, is the dried toad venom or the dried secretion from the skin glands of Bufo bufo gargarizans Cantor and has been approved to be applied in the treatment of various cancers in injection form in China [14]. The antitumor activity of Chan-su could be attributed to three cardiac glycosides including bufalin, resibufogenin, and cinobufagin. There have been researches demonstrating that bufalin, the major digoxin-like immunoreactive component of Chan-Su extracts, could trigger apoptosis and induce cell cycle arrest in many cancers including lung cancer, leukemia, gastric cancer, breast cancer, prostate cancer, melanoma, endometrial and ovarian cancer, bladder cancer, and osteosarcoma [13, 15–22]. Besides, bufalin could suppress the proliferation of colon cancer cells by inducing autophagy through promoting the reactive oxygen species and the c-Jun
Tumor Biol. (2014) 35:2461–2471
NH2-terminal kinase (JNK) signaling [14]. Results from above reports suggest that bufalin is a potential antitumor chemotherapeutic agent. However, the antitumor effect and especially the underlying molecular mechanisms have not been well understood yet in pancreatic cancer until now. Hsp27 is a member of heat shock protein family. Many studies have demonstrated that over-expression of Hsp27 plays a critical role in oncogenesis, cancer progression, and chemotherapy resistance, presumably due to its capacity to block the apoptotic process [23, 24]. Notably, the expression level of Hsp27 is constitutively up-regulated in multiple malignancies including lung cancer, gastric cancer, ovarian cancer, and acute myeloid leukemia suggesting agents that can inhibit or degrade Hsp27 may have the potential to suppress the proliferation of these cancers [25–28]. Studies concerning the exact biological role of Hsp27 in pancreatic cancer are still lacking, but it has been demonstrated by Melle et al. that the expression level of Hsp27 was up-regulated in pancreatic carcinoma tissues compared with normal pancreatic tissues. Furthermore, they reported that the concentration of Hsp27 protein in serum from cancer patients was higher than that in serum from the healthy controls, which showed the potential of Hsp27 as a serum marker for pancreatic cancer [29]. Besides, another study suggested Hsp27 as a prognostic and predictive biomarker in pancreatic ductal adenocarcinoma, which further emphasized the importance of Hsp27 in pancreatic cancer [30]. In recent years, the role of Hsp27 in the intrinsic and acquired resistance to gemcitabine of pancreatic cancer was also of interest to many researchers. In fact, Hsp27 was shown to be up-regulated in gemcitabineresistant pancreatic cancer compared with gemcitabinesensitive pancreatic cancer [31, 32]. Moreover, it is demonstrated that treatment strategies combining Hsp27 inhibitors such as KNK437, OGX-427, or interferon-γ with gemcitabine could effectively suppress the proliferation of pancreatic cancer cells [33–35]. Taken together, these results suggest that exploiting Hsp27 inhibitors alone or in combination with gemcitabine may provide novel strategies for pancreatic cancer treatment. Therefore, it is imperative to identify agents which could effectively target and inhibit Hsp27. Interestingly, a most recent study reported that bufalin could down-regulate the expression level of Hsp27 and its partner molecules (p-Akt and NF-kB) in osteosarcoma and demonstrated that Hsp27 played an important role in bufalininduced apoptosis [13]. Therefore, we are interested to identify whether bufalin could regulate the antiapoptosis protein Hsp27 in pancreatic cancer cells. In the present study, using two human pancreatic cancer cell lines PANC-1 and CFPAC-1, we found that bufalin could potentially suppress the proliferation of pancreatic cancer cells by inducing apoptosis and G2/M phase cycle arrest and sensitize them to gemcitabine-induced cell growth suppression. Further study demonstrated that bufalin could
Tumor Biol. (2014) 35:2461–2471
down-regulate the protein level of both Hsp27 and its partner molecule p-Akt and influence the expression of apoptosisrelated molecules including Bcl-2, Bax, pro-caspase-3, and pro-caspase-9. Meanwhile, the G2/M phase cell cycle arrest induced by bufalin was accompanied by down-regulation of cyclinB1 and CDK1 and up-regulation of p21 protein. Based on these data, we suggest that bufalin exerts significant antitumor effects by triggering apoptosis and inducing cell cycle arrest in pancreatic cancer cells. In addition, we speculate that bufalin-induced apoptosis in pancreatic cancer could be mediated via targeting Hsp27. This speculation and the mechanism via which bufalin caused G2/M cell cycle arrest need to be further explored.
Materials and methods Reagents and antibodies Bufalin was purchased from Cayman chemical Co. (Michigan, USA); cell counting kit-8 (CCK-8) and Annexin V/PI apoptotic detection kit were purchased from Bestbio Co. (Shanghai, China). Gemcitabine was purchased from Lily France (Fegersheim, France). Antibodies cyclinB1, CDK1, p21, caspase9, Bcl-2, Bax, and β-actin were purchased from Santa Cruz Biotechnology (Carlsbad, CA, USA). Antibodies caspase3, p-Akt, Akt, and Hsp27 were purchased from Cell Signal Technology (Danvers, MA, USA).
Cell culture and drug treatment The human pancreatic cancer PANC-1 and CFPAC-1 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Both PANC-1 and CFPAC-1 cells were cultured in RPMI 1640 medium (Hyclone China Ltd., China). All media contained 10 % FBS (Gibco, Grand Island, NY, USA), 100 μg/ml penicillin and 100 μg/ml streptomycin. All cells were cultured at 37 °C with 5 % CO2. Bufalin was dissolved in DMSO (maximum concentration, 20 mg/ml). Gemcitabine was dissolved in phosphate-buffered saline (PBS). After cancer cells were cultured overnight, the medium was changed to fresh RPMI 1640 medium and cells were exposed to bufalin for 24, 48, and 72 h before they were processed for cell growth analysis and were treated for 48 h for apoptosis and cell cycle distribution analysis. To assess the chemotherapeutic sensitization effect of bufalin, pancreatic cancer cells were treated with bufalin alone, gemcitabine alone, or their combination for 48 h, and then underwent CCK-8 analysis. For all the experiments, the cells treated with 0.1 % DMSO served as control.
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Cell viability assay The effects of bufalin on cell proliferation and the viability of the PANC-1 and CFPAC-1 cells were determined by using CCK-8. PANC-1 and CFPAC-1 cells were seeded in quadruplicate in 96-well plates at 5.0×103cells/well in a final volume of 100 μl and treated with different concentrations (50, 100, 200, 500, 1,000, 2,000 nM) of bufalin and cells treated with 0.1 % DMSO served as control. After treatment for 24, 48, and 72 h, CCK-8 (10 μl) was added to each well containing 100 μl of the culture medium, and the plate was incubated for 4 h at 37 °C. The absorbance of each well was measured at 490 nm with a microplate reader. Besides, to test whether bufalin could sensitize pancreatic cancer cells to the growth suppression effect of gemcitabine, PANC-1 and CFPAC-1 cells were seeded in quadruplicate in 96-well plates and cultured overnight. Then cells were treated with bufalin alone, gemcitabine alone, or their combination for 48 h. CCK8 assay was used to analyze the results. All experiments were repeated at least three times. Cell apoptosis detection Cell apoptosis was assessed by flow cytometry using Annexin V-FITC assay kit according to manufacturer's protocol. After being treated with different concentrations of bufalin (0–200 nM) for the indicated time (48 h), the adherent cells were washed once with PBS, harvested with trypsin, collected by centrifugation (1,000 rpm for 5 min), and washed twice with cold PBS. Cells at 4×105 were then re-suspended in Annexin V binding buffer at a final concentration of 1× 106cells/ml. Then cells were stained with 5 μl of Annexin V-FITC for 15 min and 10 μl propidium iodide (to distinguish the necrotic cells) for 5 min at 2–8 °C in the dark. After incubation, the fraction of apoptotic cells was analyzed by flow cytometry (BD FACSCalibur TM, San Jose, CA, USA) using 488 nm excitation and determined with FlowJo software (Tree Star, San Carlos, CA, USA). Flow cytometric analysis of cell cycle distribution The method has been described previously [36]. Briefly, after treatment, the cells were washed once with PBS, collected by trypsinization and centrifugation, and washed twice with icecold PBS. Cells at 1×106 per sample were fixed in 3 ml of cold 70 % ethanol at −20 °C overnight. After centrifugation (1,000 rpm for 5 min), the fixed cells were washed with PBS once and re-suspended with 500 μl of PBS containing 100 μg/ ml RNase and 5 μg/ml propidium iodide and incubated at room temperature in the dark for 30 min. Flow cytometric analysis was performed using FACScan (BD FACSCalibur TM, San Jose, CA, USA) for determining the percentage of
2464
Tumor Biol. (2014) 35:2461–2471
cells in various phases of the cell cycle. At least 10,000 events were counted. Western blotting After being exposed to different concentrations of bufalin (0– 200 nM) for 48 h, the pancreatic cancer cells were collected and lysed. Protein concentration was determined using BCA protein assay (Beyotime Institute of Biotechnology, Beijing, China) according to the manufacturer' instructions. For each sample, about 40 μg of protein was separated by SDS-PAGE (10 %) and electro-transferred onto PVDF membrane. The membrane was blocked in 5 % skim milk in Tris-buffered saline with Tween 20 (TBST) buffer for 1 h and then incubated with individual primary antibodies overnight at 4 °C. The membrane was washed by TBST buffer for three times and then incubated with secondary antibody-conjugated horseradish peroxidase for 1 h at room temperature. The protein bands on the membrane could be visualized on Xray film using the enhanced chemiluminescence (ECL) kit. The protein levels were normalized by β-actin. Statistical analysis Data obtained from this research were analyzed by Student's t test or one-way ANOVA, and a p value of 0.05 was considered as significant. All analysis was performed by SPSS 17.0.
Results Bufalin inhibits the proliferation of pancreatic cancer cells The structure of bufalin, a major digoxin-like immunoreactive component of Chan-Su extracts, is shown in Fig. 1a. We first tested the cytotoxic efficacy of bufalin on two pancreatic cancer cell lines (PANC-1 and CFPAC-1) by using CCK-8 analysis. After treatment with different concentrations (0– 2,000 nM) of bufalin for 24, 48, and 72 h, the proliferation of pancreatic cancer cells was significantly suppressed in a dose- and time-dependent manner, as shown in Fig. 1b. Bufalin induces apoptosis and alters the level of apoptosis-related proteins in pancreatic cancer cells Inhibition of cell proliferation is usually caused by cell death and cell cycle arrest. According to results from the CCK-8 assay in which we tested the efficacy of bufalin on the growth of pancreatic cancer cells, we selected the doses of 50, 100, and 200 nM of bufalin to explore the underlying mechanism of bufalin-induced proliferation inhibition of the cells. After treatment with different concentrations of bufalin (0–200 nM) for 48 h, pancreatic cancer cells (PANC-1 and CFPAC-1 cells)
Fig. 1 Effects of bufalin on cell proliferation in human pancreatic cancer cells. a The chemical structure of bufalin. b PANC-1 and CFPAC-1 cells were treated with different concentrations of bufalin (0–2,000 nM) for 24, 48, and 72 h. Cell viability was measured by CCK-8 analysis. Data shown are representative of at least three independent experiments. *P
RESEARCH ARTICLE
Bufalin exerts antitumor effects by inducing cell cycle arrest and triggering apoptosis in pancreatic cancer cells Meiying Li & Xuejun Yu & Hui Guo & Limei Sun & Aijun Wang & Qiji Liu & Xiuwen Wang & Jisheng Li
Received: 6 August 2013 / Accepted: 14 October 2013 / Published online: 12 November 2013 # International Society of Oncology and BioMarkers (ISOBM) 2013
Abstract As one of the most aggressive human malignancies, pancreatic cancer is a leading cause of cancer-related deaths worldwide and only about 4 % of patients will live 5 years after diagnosis. Eighty to approximately eighty-five percent of patients are diagnosed with an unresectable or metastatic disease, which is correlated with poor prognosis and low survival rate. Therefore, it is tremendously significant to exploit novel chemicals to prevent and treat pancreatic cancer. Previous research and clinical studies have demonstrated that many natural products derived from traditional Chinese medicine (TCM) such as camptothecin derivatives and vinca alkaloids could be effective antitumor compounds, hinting that TCM is a promising source for developing new antitumor drugs. In this report, we investigated the effects of bufalin, a primary active ingredient of the traditional Chinese medicine Chan-Su, on pancreatic cancer cell lines PANC-1 and CFPAC1 and studied the underlying molecular mechanism. We found that exposure to bufalin could suppress the proliferation of pancreatic cancer cells time and dose dependently. We used flow cytometry to study the effects of bufalin on apoptosis and cell cycle distribution in PANC-1 and CFPAC-1 cells. The results indicated that bufalin could significantly induce both
apoptosis and G2/M cell cycle arrest in pancreatic cancer cells. With western blotting, we found that the expression level of an antiapoptotic protein heat shock protein 27 (Hsp27) and its partner molecule p-Akt was decreased upon the treatment with bufalin. Besides, bufalin activated pro-caspase-3 and procaspase-9 and modulated the expression level of Bcl-2 and Bax. These data suggested that bufalin may trigger apoptosis by targeting Hsp27, which could inhibit apoptosis by interfering with key apoptotic proteins. The influence on the level of cylinB1, CDK1, and p21 was also observed after bufalin treatment, and the relationship between Hsp27 and the cell cycle-related proteins mentioned above deserves much more research. In addition, our data showed that bufalin could enhance the growth inhibition effect of gemcitabine in above pancreatic cancer cells. Taken together, bufalin might be worthy of further study for its potential as a therapeutic agent for pancreatic cancer treatment. Keywords Pancreatic cancer . Bufalin . Antitumor . Cell cycle arrest . Apoptosis . Hsp27
Introduction Meiying Li and Xuejun Yu contributed equally to this work. M. Li : X. Yu : L. Sun : A. Wang : X. Wang (*) : J. Li (*) Department of Medical Oncology, Cancer Center, Qilu Hospital, Shandong University, Jinan 250012, China e-mail: [email protected] e-mail: [email protected] H. Guo Department of Ophthalmology, Qilu Hospital of Shandong University, Jinan 250012, China Q. Liu Department of Medical Genetics, Key Laboratory for Experimental Teratology of the Ministry of Education, Shandong University School of Medicine, Jinan 250012, China
Pancreatic cancer, one of the most aggressive and fatal human malignancies, is currently the fourth leading cause of cancer death in the USA and leads to about 227,000 deaths per year worldwide [1]. In China, pancreatic cancer is the sixth leading cause of cancer-related deaths, and the 5-year survival rate is only 1 to 3 % [2]. Despite advancements of the diagnosis and management of pancreatic cancer, the incidence and mortality of this malignancy have kept increasing in recent years. It is estimated that about 45,220 new cases of pancreatic cancer will be diagnosed in the USA in 2013, and 38,460 patients will die of this disease [3]. The poor prognosis of pancreatic cancer is mainly attributed to its hallmarks including delayed clinical
2462
presentation, tendency of early local and distant dissemination, and resistance to conventional chemo-radiation treatment strategies [4]. At present, surgical resection offers the only chance of cure for those diagnosed with cancer localized to the pancreas, and adjuvant chemotherapy after surgery is recommended to delay relapse and improve survival rate. Unfortunately, up to 85 % of patients present with advanced unresectable disease and the available effective regimens are quite limited [1]. For patients with advanced and metastatic pancreatic cancer, chemotherapy is the primary treatment modality and gemcitabine is routinely used for alleviating patients' symptoms since 1997 [5]. Besides monotherapy, gemcitabine has been investigated in combination with synergistic agents such as cisplatin, capecitabine, and erlotinib [6–9]. In addition, the regimen of FOLFIRINOX (5fluorouracil, oxaliplatin, irinotecan, and leucovorin) has shown superiority over gemcitabine monotherapy in prolonging patients' overall survival, but it is only limited for patients with good performance status [10]. Recently, the results of MPACT study showed that the median overall survival of patients who received nab-paclitaxel plus gemcitabine was 8.5 months, compared with 6.7 months for those who received gemcitabine monotherapy [11]. This is a real breakthrough in pancreatic cancer treatment. Despite above advancements in the chemotherapy of pancreatic cancer, gemcitabine still remains as the standard regimen for treating pancreatic cancer. However, only part of patients are responsive to gemcitabine treatment because of the inherent and acquired drug resistance and the adverse events could be intolerable for some of them [5, 12]. Therefore, it is urgent to identify novel effective anticancer drug to treat this deadly malignancy. Poisonous Chinese herbal medicine (PCHM) which is derived from botanicals, minerals, and animals has been historically used in cancer therapies by skilled Chinese practitioners for thousands of years. Numerous researches mining novel anticancer drugs from PCHM-derived natural products proved that PCHM is a huge and noteworthy reservoir for novel efficacious anticancer agents [13]. ChanSu, one such Chinese herbal medicine, is the dried toad venom or the dried secretion from the skin glands of Bufo bufo gargarizans Cantor and has been approved to be applied in the treatment of various cancers in injection form in China [14]. The antitumor activity of Chan-su could be attributed to three cardiac glycosides including bufalin, resibufogenin, and cinobufagin. There have been researches demonstrating that bufalin, the major digoxin-like immunoreactive component of Chan-Su extracts, could trigger apoptosis and induce cell cycle arrest in many cancers including lung cancer, leukemia, gastric cancer, breast cancer, prostate cancer, melanoma, endometrial and ovarian cancer, bladder cancer, and osteosarcoma [13, 15–22]. Besides, bufalin could suppress the proliferation of colon cancer cells by inducing autophagy through promoting the reactive oxygen species and the c-Jun
Tumor Biol. (2014) 35:2461–2471
NH2-terminal kinase (JNK) signaling [14]. Results from above reports suggest that bufalin is a potential antitumor chemotherapeutic agent. However, the antitumor effect and especially the underlying molecular mechanisms have not been well understood yet in pancreatic cancer until now. Hsp27 is a member of heat shock protein family. Many studies have demonstrated that over-expression of Hsp27 plays a critical role in oncogenesis, cancer progression, and chemotherapy resistance, presumably due to its capacity to block the apoptotic process [23, 24]. Notably, the expression level of Hsp27 is constitutively up-regulated in multiple malignancies including lung cancer, gastric cancer, ovarian cancer, and acute myeloid leukemia suggesting agents that can inhibit or degrade Hsp27 may have the potential to suppress the proliferation of these cancers [25–28]. Studies concerning the exact biological role of Hsp27 in pancreatic cancer are still lacking, but it has been demonstrated by Melle et al. that the expression level of Hsp27 was up-regulated in pancreatic carcinoma tissues compared with normal pancreatic tissues. Furthermore, they reported that the concentration of Hsp27 protein in serum from cancer patients was higher than that in serum from the healthy controls, which showed the potential of Hsp27 as a serum marker for pancreatic cancer [29]. Besides, another study suggested Hsp27 as a prognostic and predictive biomarker in pancreatic ductal adenocarcinoma, which further emphasized the importance of Hsp27 in pancreatic cancer [30]. In recent years, the role of Hsp27 in the intrinsic and acquired resistance to gemcitabine of pancreatic cancer was also of interest to many researchers. In fact, Hsp27 was shown to be up-regulated in gemcitabineresistant pancreatic cancer compared with gemcitabinesensitive pancreatic cancer [31, 32]. Moreover, it is demonstrated that treatment strategies combining Hsp27 inhibitors such as KNK437, OGX-427, or interferon-γ with gemcitabine could effectively suppress the proliferation of pancreatic cancer cells [33–35]. Taken together, these results suggest that exploiting Hsp27 inhibitors alone or in combination with gemcitabine may provide novel strategies for pancreatic cancer treatment. Therefore, it is imperative to identify agents which could effectively target and inhibit Hsp27. Interestingly, a most recent study reported that bufalin could down-regulate the expression level of Hsp27 and its partner molecules (p-Akt and NF-kB) in osteosarcoma and demonstrated that Hsp27 played an important role in bufalininduced apoptosis [13]. Therefore, we are interested to identify whether bufalin could regulate the antiapoptosis protein Hsp27 in pancreatic cancer cells. In the present study, using two human pancreatic cancer cell lines PANC-1 and CFPAC-1, we found that bufalin could potentially suppress the proliferation of pancreatic cancer cells by inducing apoptosis and G2/M phase cycle arrest and sensitize them to gemcitabine-induced cell growth suppression. Further study demonstrated that bufalin could
Tumor Biol. (2014) 35:2461–2471
down-regulate the protein level of both Hsp27 and its partner molecule p-Akt and influence the expression of apoptosisrelated molecules including Bcl-2, Bax, pro-caspase-3, and pro-caspase-9. Meanwhile, the G2/M phase cell cycle arrest induced by bufalin was accompanied by down-regulation of cyclinB1 and CDK1 and up-regulation of p21 protein. Based on these data, we suggest that bufalin exerts significant antitumor effects by triggering apoptosis and inducing cell cycle arrest in pancreatic cancer cells. In addition, we speculate that bufalin-induced apoptosis in pancreatic cancer could be mediated via targeting Hsp27. This speculation and the mechanism via which bufalin caused G2/M cell cycle arrest need to be further explored.
Materials and methods Reagents and antibodies Bufalin was purchased from Cayman chemical Co. (Michigan, USA); cell counting kit-8 (CCK-8) and Annexin V/PI apoptotic detection kit were purchased from Bestbio Co. (Shanghai, China). Gemcitabine was purchased from Lily France (Fegersheim, France). Antibodies cyclinB1, CDK1, p21, caspase9, Bcl-2, Bax, and β-actin were purchased from Santa Cruz Biotechnology (Carlsbad, CA, USA). Antibodies caspase3, p-Akt, Akt, and Hsp27 were purchased from Cell Signal Technology (Danvers, MA, USA).
Cell culture and drug treatment The human pancreatic cancer PANC-1 and CFPAC-1 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Both PANC-1 and CFPAC-1 cells were cultured in RPMI 1640 medium (Hyclone China Ltd., China). All media contained 10 % FBS (Gibco, Grand Island, NY, USA), 100 μg/ml penicillin and 100 μg/ml streptomycin. All cells were cultured at 37 °C with 5 % CO2. Bufalin was dissolved in DMSO (maximum concentration, 20 mg/ml). Gemcitabine was dissolved in phosphate-buffered saline (PBS). After cancer cells were cultured overnight, the medium was changed to fresh RPMI 1640 medium and cells were exposed to bufalin for 24, 48, and 72 h before they were processed for cell growth analysis and were treated for 48 h for apoptosis and cell cycle distribution analysis. To assess the chemotherapeutic sensitization effect of bufalin, pancreatic cancer cells were treated with bufalin alone, gemcitabine alone, or their combination for 48 h, and then underwent CCK-8 analysis. For all the experiments, the cells treated with 0.1 % DMSO served as control.
2463
Cell viability assay The effects of bufalin on cell proliferation and the viability of the PANC-1 and CFPAC-1 cells were determined by using CCK-8. PANC-1 and CFPAC-1 cells were seeded in quadruplicate in 96-well plates at 5.0×103cells/well in a final volume of 100 μl and treated with different concentrations (50, 100, 200, 500, 1,000, 2,000 nM) of bufalin and cells treated with 0.1 % DMSO served as control. After treatment for 24, 48, and 72 h, CCK-8 (10 μl) was added to each well containing 100 μl of the culture medium, and the plate was incubated for 4 h at 37 °C. The absorbance of each well was measured at 490 nm with a microplate reader. Besides, to test whether bufalin could sensitize pancreatic cancer cells to the growth suppression effect of gemcitabine, PANC-1 and CFPAC-1 cells were seeded in quadruplicate in 96-well plates and cultured overnight. Then cells were treated with bufalin alone, gemcitabine alone, or their combination for 48 h. CCK8 assay was used to analyze the results. All experiments were repeated at least three times. Cell apoptosis detection Cell apoptosis was assessed by flow cytometry using Annexin V-FITC assay kit according to manufacturer's protocol. After being treated with different concentrations of bufalin (0–200 nM) for the indicated time (48 h), the adherent cells were washed once with PBS, harvested with trypsin, collected by centrifugation (1,000 rpm for 5 min), and washed twice with cold PBS. Cells at 4×105 were then re-suspended in Annexin V binding buffer at a final concentration of 1× 106cells/ml. Then cells were stained with 5 μl of Annexin V-FITC for 15 min and 10 μl propidium iodide (to distinguish the necrotic cells) for 5 min at 2–8 °C in the dark. After incubation, the fraction of apoptotic cells was analyzed by flow cytometry (BD FACSCalibur TM, San Jose, CA, USA) using 488 nm excitation and determined with FlowJo software (Tree Star, San Carlos, CA, USA). Flow cytometric analysis of cell cycle distribution The method has been described previously [36]. Briefly, after treatment, the cells were washed once with PBS, collected by trypsinization and centrifugation, and washed twice with icecold PBS. Cells at 1×106 per sample were fixed in 3 ml of cold 70 % ethanol at −20 °C overnight. After centrifugation (1,000 rpm for 5 min), the fixed cells were washed with PBS once and re-suspended with 500 μl of PBS containing 100 μg/ ml RNase and 5 μg/ml propidium iodide and incubated at room temperature in the dark for 30 min. Flow cytometric analysis was performed using FACScan (BD FACSCalibur TM, San Jose, CA, USA) for determining the percentage of
2464
Tumor Biol. (2014) 35:2461–2471
cells in various phases of the cell cycle. At least 10,000 events were counted. Western blotting After being exposed to different concentrations of bufalin (0– 200 nM) for 48 h, the pancreatic cancer cells were collected and lysed. Protein concentration was determined using BCA protein assay (Beyotime Institute of Biotechnology, Beijing, China) according to the manufacturer' instructions. For each sample, about 40 μg of protein was separated by SDS-PAGE (10 %) and electro-transferred onto PVDF membrane. The membrane was blocked in 5 % skim milk in Tris-buffered saline with Tween 20 (TBST) buffer for 1 h and then incubated with individual primary antibodies overnight at 4 °C. The membrane was washed by TBST buffer for three times and then incubated with secondary antibody-conjugated horseradish peroxidase for 1 h at room temperature. The protein bands on the membrane could be visualized on Xray film using the enhanced chemiluminescence (ECL) kit. The protein levels were normalized by β-actin. Statistical analysis Data obtained from this research were analyzed by Student's t test or one-way ANOVA, and a p value of 0.05 was considered as significant. All analysis was performed by SPSS 17.0.
Results Bufalin inhibits the proliferation of pancreatic cancer cells The structure of bufalin, a major digoxin-like immunoreactive component of Chan-Su extracts, is shown in Fig. 1a. We first tested the cytotoxic efficacy of bufalin on two pancreatic cancer cell lines (PANC-1 and CFPAC-1) by using CCK-8 analysis. After treatment with different concentrations (0– 2,000 nM) of bufalin for 24, 48, and 72 h, the proliferation of pancreatic cancer cells was significantly suppressed in a dose- and time-dependent manner, as shown in Fig. 1b. Bufalin induces apoptosis and alters the level of apoptosis-related proteins in pancreatic cancer cells Inhibition of cell proliferation is usually caused by cell death and cell cycle arrest. According to results from the CCK-8 assay in which we tested the efficacy of bufalin on the growth of pancreatic cancer cells, we selected the doses of 50, 100, and 200 nM of bufalin to explore the underlying mechanism of bufalin-induced proliferation inhibition of the cells. After treatment with different concentrations of bufalin (0–200 nM) for 48 h, pancreatic cancer cells (PANC-1 and CFPAC-1 cells)
Fig. 1 Effects of bufalin on cell proliferation in human pancreatic cancer cells. a The chemical structure of bufalin. b PANC-1 and CFPAC-1 cells were treated with different concentrations of bufalin (0–2,000 nM) for 24, 48, and 72 h. Cell viability was measured by CCK-8 analysis. Data shown are representative of at least three independent experiments. *P
