Biochemical Pharmacology 88 (2014) 322–333
Contents lists available at ScienceDirect
Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm
Shikonin suppresses tumor growth and synergizes with gemcitabine in a pancreatic cancer xenograft model: Involvement of NF-kB signaling pathway Yongwei Wang a, Yinan Zhou a, Guang Jia a, Bing Han a, Ji Liu a, Yueqiu Teng b, Jiachen Lv a, Zengfu Song a, Yilong Li a, Liang Ji a, Shangha Pan a, Hongchi Jiang a, Bei Sun a,* a b
Department of Pancreatic and Biliary Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin 150001, China Department of Central Laboratory of Blood Cancer, The First Affiliated Hospital of Harbin Medical University, Harbin 150001, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 December 2013 Received in revised form 28 January 2014 Accepted 30 January 2014 Available online 9 February 2014
Although gemcitabine is currently the best chemotherapeutic agent available for the treatment of advanced pancreatic cancer, eventual failure of response is a significant clinical problem. Therefore, novel therapeutic approaches against this disease are highly needed. The aim of this study was to evaluate whether shikonin, a naphthoquinone derivative, has potential in the treatment of pancreatic cancer when used either alone or in combination with gemcitabine. Our in vitro results showed that shikonin inhibited the proliferation of three different human pancreatic cancer cell lines and potentiated the cytotoxic effect of gemcitabine, which correlated with the down-regulation of constitutive as well as gemcitabine-induced activation of NF-kB and NF-kB-regulated gene products. Most importantly, using a xenograft model of human pancreatic cancer, we found shikonin alone significantly suppressed tumor growth and argumented the antitumor activity of gemcitabine. These effects also correlated with the down-regulation of NF-kB activity and its target genes, decreased proliferation (PCNA and Ki-67), decreased microvessel density (CD31), and increased apoptosis (TUNEL) in tumor remnants. Collectively, our results suggest that shikonin can suppress the growth of human pancreatic tumors and potentiate the antitumor effects of gemcitabine through the suppression of NF-kB and NF-kB-regulated gene products. ß 2014 Elsevier Inc. All rights reserved.
Keywords: Shikonin Gemcitabine Pancreatic Cancer NF-kB
1. Introduction Pancreatic cancer remains one of the most fatal human malignancies and represents the fourth leading cause of cancer mortality in western countries, which has an alarmingly low 5year survival rate with best treatments available today of less than 5% [1,2]. The National Cancer Institute estimated that 45,220 men and women in the United States were diagnosed with pancreatic cancer in 2013 and 38,460 died of the disease [2]. Gemcitabine, erlotinib and abraxane are the only FDA-approved standard therapies for the treatment of patients with pancreatic cancer. However, they produce a poor response rate and are associated with various side effects and the development of chemoresistance, which exactly offsets the marginal benefit. Hence, the need to identify novel strategies against this fatal disease is imperative. Multiple lines of evidence suggest that the activation of master transcription factor NF-kB and NF-kB-regulated gene products * Corresponding author. E-mail address: [email protected] (B. Sun). http://dx.doi.org/10.1016/j.bcp.2014.01.041 0006-2952/ß 2014 Elsevier Inc. All rights reserved.
have a defined central role in growth, invasion, metastasis, angiogenesis, antiapoptosis and chemoresistance of pancreatic cancer [3–5]. First, NF-kB is constitutively active in pancreatic cancer and correlates with tumor progression and poor prognosis [6,7]. Second, it has the ability to promote pancreatic cancer growth partly via inhibition of apoptosis [8]. Third, NF-kB provides a direct stimulus toward cellular proliferation through the induction of growth promoting genes such as c-Myc and cyclin-D1, the latter of which has been shown to be overexpressed in human pancreatic cancer cell lines and cancer tissues and to have a significant inverse correlation with patient survival [9,10]. Fourth, NF-kB plays a pivotal role in the enhanced angiogenic potential of pancreatic cancer cells via increasing the production of proangiogenic factors such as VEGF [11]. Fifth, activation of NFkB is clearly associated with the migration and invasion of pancreatic cancer cells [12]. Finally, NF-kB contributes to the chemoresistance of pancreatic cancer cells [13]. Together, this evidence implicates the involvement of NF-kB pathway in pancreatic cancer and suggests that agents that can interfere with activation of NF-kB have enormous potential to suppress
Y. Wang et al. / Biochemical Pharmacology 88 (2014) 322–333
323
Fig. 1. Effect of shikonin on cell viability. (A) Chemical structure of shikonin. (B) Cell viability assay. Three pancreatic cancer cells (BxPC-3, PANC-1 and AsPC-1) and normal rat skeletal muscle cells (L6) were treated with varying concentrations of shikonin for 24, 48 and 72 h and cell viability was measured by CCK-8 assay. Data shown are representative of at least three independent experiments. * P < 0.05; ** P < 0.01 versus control.
tumor growth and may also serve to improve the effectiveness of gemcitabine. Shikonin (Fig. 1A), a natural naphthoquinone, is one such agent. Derived from the Chinese medical herb Lithospermum erythrorhizon, shikonin has been widely used as a traditional Chinese medicine for thousands of years to treat sore throats, macular eruptions, measles, carbuncles and burns [14,15]. Recently, emerging evidence suggests that shikonin has high efficacy against a series of human cancer cell lines in vitro and in several animal models in vivo with minimal or no toxicity to nonmalignant human cells [16–22]. The antitumor effect of shikonin may be associated with its ability to inhibit the expression of anti-apoptotic BCL-2 family members [23], activate caspases [24,25], decrease proteasome activity [22], suppress EGFR phosphorylation [26], generate reactive oxygen species (ROS) [27,28], arrest the cell cycle through p53 upregulation [29], activate the stress-related c-Jun-N-terminal kinase (JNK) pathway [30], and inactivate Akt and NF-kB pathways [21,31]. Collectively, these scientific findings suggest that shikonin may have a strong potential for future use in cancer prevention and treatment. However, the effect of shikonin on the growth of pancreatic cancer is not known. It is also not known whether shikonin has potential to act as a chemosensitizing agent to improve the therapeutic index of gemcitabine in pancreatic cancer cell lines and xenograft models.
In the present study, we investigated whether shikonin alone could suppress the growth of human pancreatic cancer cells both in vitro and in a xenograft mouse model, and whether shikonin could enhance chemosensitivity to gemcitabine. We found that shikonin inhibited the proliferation of various pancreatic cancer cells, potentiated gemcitabine-induced growth inhibition and apoptosis in pancreatic cancer cells in vitro and significantly enhanced the antitumor effects of gemcitabine in human xenograft pancreatic cancer model through the modulation of NF-kB and NF-kB-linked gene products.
2. Materials and methods 2.1. Materials Shikonin was purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Gemcitabine (Gemzar) was purchased from Eli Lilly. Antibodies used in this study included Antibodies against Survivin, procaspase-3, procaspase-9, c-Myc, Bcl-2, Bcl-xL, VEGF, PCNA and XIAP (Santa Cruz Biotechnology, CA, USA), Antibodies against Ki-67 and CD31 (Abcam Inc., MA, USA), and Antibodies against p-IkB-a, IkB-a, cyclin D1, COX-2, PARP (Cell Signaling Technology, Inc., MA, USA). Nuclear Extract Kit and Trans-AM NF-kB p65 ELISA Kit were obtained from Active Motif (Carlsbad, CA, USA).
324
Y. Wang et al. / Biochemical Pharmacology 88 (2014) 322–333
2.2. Treatment of cells Shikonin dissolved in DMSO [final concentration, 0.1% (v/v)] was used for the treatment of cells. The subconfluent cells (60–70% confluent) were treated with various concentrations of shikonin alone, gemcitabine alone or their combinations (as indicated in the figure legends) in complete cell culture medium and cells treated with vehicle (DMSO) served as control. The following experiments were repeated thrice. 2.3. Cell culture The pancreatic cancer cell lines PANC-1, BxPC-3 and AsPC-1 were obtained from the American Type Culture Collection (Manassas, VA). Rat normal skeletal muscle cell lines L6 were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China. PANC-1 and L-6 were cultured in DMEM and BxPC-3 and AsPC-1 was cultured in RPMI 1640. All media were supplemented with fetal bovine serum (10%), penicillin (100 U/ml) and streptomycin (100 mg/ml) (Irvine ScientiWc, Irvine, CA) and maintained at 37 8C in humidified air with 5% CO2 (all reagents were from HyClone China Ltd., China). 2.4. Cell viability assay The viability of treated cells was determined by using Cell Counting Kit-8 (CCK-8) kit (Dojindo Laboratories, Kumamoto, Japan) following the instructions outlined by the manufacturer and as previously described by us [32]. 2.5. Combination index analysis To evaluate the synergistic efficacies of shikonin and gemcitabine, the combination index (CI) isobologram method of Chou and Talalay was used. This commonly used analysis involves plotting concentration–effect curves for each agent and for multiply diluted fixed-ratio combinations by using the medianeffect equation and the combination index equation. A value of CI less than, equal to or greater than 1 indicates synergism, additivity and antagonism, respectively. The combination index values were calculated at multiple effect levels and the isobolograms plotted. 2.6. Apoptosis assay Apoptosis was evaluated by using (i) Cell Death Detection ELISA kit (Roche, Palo Alto, CA, USA) or (ii) Annexin V-FITC kit (BD Biosciences, San Jose, California, USA) following the instructions outlined by the manufacturer. 2.7. Caspase activity assay Caspase-3 and caspase-9 activities were measured using the Caspase Activity Kit (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China) following the instructions outlined by the manufacturer and as previously described by us [32]. 2.8. Western blot analysis To determine the levels of protein expression, cell lysates were prepared and subjected to Western blot analysis as described previously [32].
at 37 8C. The cells were then transfected with NF-kB p65 cDNA, control empty vector (pcDNA3.0), NF-kB p65 siRNA and control siRNA, respectively, using Lipofectamine 2000 (Invitrogen). The transfections were performed following the instructions outlined by the manufacturer. After transfection, cells were treated as designed before being harvested for the following studies. 2.10. Xenograft mouse model Animal studies were in accordance with the institutional guide for the care and use of laboratory animals. Our experimental protocol was reviewed and approved by the Animal Care and Use Committee of Harbin Medical University (Harbin, Heilongjiang, China). Male nude BALB/c mice (4–6 weeks old) were obtained from Shanghai Experimental Animal Center of the Chinese Academy of Sciences. Tumors were established by subcutaneous injection of 5 106 BxPC-3 cells into the flanks of mice. Tumor size was measured every three days with calipers and the volume was estimated according to the formula: V = (p/6) (larger diameter) (smaller diameter)2. When tumors reached around 100 mm3, the mice were randomly assigned to four groups (each group had eight mice): (a) control (DMSO was dissolved in 200 ml PBS, once daily by i.p. injection); (b) shikonin (2 mg/kg, once daily by i.p. injection); (c) gemcitabine (100 mg/kg, twice weekly by i.p. injection); and (d) shikonin and gemcitabine, following the same schedule of individual drugs. The doses of shikonin and gemcitabine selected for this experiment were based on preliminary experiments and previous studies. The mice were closely monitored for 24 days, then euthanized, and the tumors were removed. Each tumor was divided into two pieces: one fixed in 10% buffered formalin, and the other kept at 80 8C for further analysis. 2.11. PCNA and Ki-67 immunohistochemistry The methodology has been described previously [32]. In brief, paraffin-embedded tissue sections (5 mm) were immunostained with an anti-PCNA or anti-Ki-67 Ab. The number of PCNA or Ki-67 positive cells was counted in randomly selected ten microscopic fields at 400 magnification. 2.12. Quantification of apoptosis in tumor sections The methodology has been described previously [32]. In brief, paraffin-embedded tissue sections (5 mm) were stained with the TUNEL agent (Roche, Shanghai, China), and the number of TUNELpositive cells was counted in randomly selected 10 microscopic fields at 400 magnification. 2.13. Tumor microvessel density The methodology has been described previously [34]. In brief, tumor sections prepared from tumors 24 days after treatment were immunostained with anti-CD31 Ab. The number of microvessels was counted in randomly selected ten microscopic fields at 400 magnification, and the microvessel density was recorded. 2.14. Immunohistochemical analysis for cyclin D1, MMP-9, and VEGF in tumor samples The expression of cyclin D1, MMP-9, and VEGF were evaluated by immunohistochemical analysis as described previously [34]. 2.15. NF-kB DNA-binding activity assay
2.9. Plasmids and transfections The methodology has been described previously [33]. In brief, BxPC-3 cells were seeded in six-well plates and incubated for 24 h
The NF-kB DNA-binding activity was determined using the Trans-Am NF-kB/p65 ELISA Kit following the instructions outlined by the manufacturer and as described previously [35].
Y. Wang et al. / Biochemical Pharmacology 88 (2014) 322–333
2.16. Statistical analysis Data are represented as mean values standard deviation (SD). Statistical comparisons were made with a one-way analysis of variance (ANOVA) followed by Dunnett’s t test for multiple comparisons. A P value of less than 0.05 was considered significant.
3. Results The objective of this study was, first, to determine whether shikonin has potential in the treatment of pancreatic cancer when used either alone or in combination with gemcitabine and, second, to explore the molecular mechanism(s) behind these effects. Three different human pancreatic cancer cell lines (BxPC-3, PANC-1 and AsPC-1) of different origin were selected for this investigation. To monitor tumor growth in vivo, one of these cell lines, BxPC-3 was subcutaneously injected and used in a mouse xenograft model. 3.1. Effect of shikonin on cell viability To investigate the effect of shikonin on the proliferation of human pancreatic cancer cells, we used BxPC-3, PANC-1, and AsPC-1 cells, all of which exhibit differential resistance pattern to conventional chemotherapeutic drugs. The cells were assessed for viability using a CCK-8 assay following exposure to increasing concentrations of shikonin (0–10 mM) for 24, 48, or 72 h. As shown in Fig. 1B, shikonin suppressed the growth of all three human pancreatic cancer cells in a dose- and time-dependent manner. These data suggest that shikonin was an effective inhibitor of pancreatic cancer cell proliferation as a single agent. However, shikonin had minimal effect on normal rat skeletal muscle cells (Fig. 1B). 3.2. Shikonin potentiates growth inhibition caused by gemcitabine in pancreatic cancer cells We investigated the effects of shikonin and gemcitabine, alone or in combination, on cell viability by CCK-8 assay. For these studies, cells were pretreated with shikonin or left untreated as control for 24 h before incubating with or without gemcitabine for 48 h, and cell viability was determined at 72 h post-treatment. The doses of shikonin and gemcitabine used here were chosen based on our preliminary dose escalation studies and previous reports. As shown in Fig. 2A, treatment with either shikonin for 72 h or gemcitabine alone for 48 h resulted in only 25% to 40% growth inhibition in pancreatic cancer cells. However, pretreatment with shikonin for 24 h followed by treatment with gemcitabine for 48 h resulted in 65% to 80% growth inhibition in all three pancreatic cancer cell lines studied. The nature of the interaction between shikonin and gemcitabine was calculated by using the Chou/Talalay median-effect equation method for determining the combination index. A CI < 1 indicates synergy, and the CI range values for the combination of shikonin and gemcitabine in three different human pancreatic cancer cell lines were 0.4 to 0.9 for fractional effect corresponding to 0.3 to 0.9 (Fig. 2B), indicating that shikonin in combination with gemcitabine produces synergistic cell cytotoxicity in all three pancreatic cancer cells tested. These results show that shikonin in combination with low therapeutic doses of gemcitabine elicits significantly more loss of cell viability of pancreatic cancer cells than either agent alone, suggesting that therapeutic approaches devised for better cancer cell killing and fewer harmful side effects may be formulated. 3.3. Shikonin potentiates gemcitabine-induced apoptosis in human pancreatic cancer cells We next confirmed whether enhanced cytotoxicity by combination treatment of shikonin and gemcitabine was due
325
to induction of apoptosis. By histone DNA ELISA and Annexin V/ FITC flow cytometric analysis, we observed similar results showing that treatment with either shikonin or gemcitabine alone induced apoptosis in all three cell lines tested. Relative to single agents, shikonin pretreatment followed by gemcitabine treatment led to significantly higher apoptosis in the cancer cells (Fig. 2C and D). These results are consistent with cell viability assay, suggesting that greater cell growth inhibition resulting from combination treatment with shikonin and gemcitabine may due, at least partly, to the induction of more apoptosis in pancreatic cancer cells. 3.4. Molecular basis for shikonin and gemcitabine augmenting signal for apoptosis In an attempt to explore the detailed molecular mechanism(s) of shikonin-induced apoptosis and chemosensitization, we proceeded to assess the expression levels of procaspase-3, procaspase9 and cleavage of PARP, and modulation of the status of other apoptosis-related proteins. Western blotting revealed that both shikonin and gemcitabine alone down-regulated the expression of procaspase-3 and procaspase-9 in all three human pancreatic cancer cells and the combination was more effective (Fig. 2E). These findings were further confirmed by caspase activity assays showing that caspase-3 and caspase-9 activities were significantly increased by combination treatment with shikonin and gemcitabine (Fig. 2F). Since PARP is a substrate for caspase activity and a reliable marker of apoptosis, we next examined the level of cleaved PARP. Our data showed that whereas treatment of BxPC-3, PANC-1 and AsPC-1 cells with single agents showed low levels of PARP cleavage; shikonin pretreatment followed by gemcitabine treatment resulted in a stronger PARP cleavage (Fig. 2E). Furthermore, experiments were done to examine the expression levels of other apoptosis-related proteins. As shown in Fig. 3D, the level of c-Myc, COX-2, cyclin D1, Survivin, Bcl-2, Bcl-XL, XIAP, MMP-9 and VEGF were significantly decreased in the combination treatment group compared with control or single agent-treated group. Together, these results provide insight into the mechanism by which shikonin exerts its effects in pancreatic cancer cells. Since all of the above-mentioned proteins are known to be regulated by NF-kB, we examined whether shikonin exerts its effects by interfering with the NF-kB pathway. 3.5. Shikonin inhibits constitutive as well as gemcitabine-induced activation of NF-kB in pancreatic cancer cells Because NF-kB has been associated with both proliferation and apoptotic resistance, we next examined the effects of shikonin, gemcitabine, and the combination on NF-kB activity using an ELISA-based DNA-binding assay. Consistent with previous studies [32,34,36], gemcitabine alone was able to induce NF-kB DNA-binding activity (Fig. 3C). In addition, our results showed that NF-kB p65 siRNA transfection significantly inhibited gemcitabine-induced NF-kB DNA-binding activity whereas NF-kB p65 cDNA transfection enhanced activation of NF-kB DNA-binding activity stimulated by gemcitabine (Fig. 4A and B). As shown in Fig. 3A and B, we further found that treatment with shikonin caused a concentration-dependent decrease in NF-kB DNA-binding activity in all three pancreatic cancer cells, which was associated with a concentrationdependent decrease in phospho-IkB-a with a concomitant concentration-dependent increase in the cytosolic levels of the IkB-a protein. More importantly, shikonin was also found to significantly suppress gemcitabine- or p65 cDNA transfectioninduced activation of NF-kB DNA-binding activity (Fig. 3C and
326
Y. Wang et al. / Biochemical Pharmacology 88 (2014) 322–333
Fig. 2. Effect of shikonin and gemcitabine on cell viability and apoptosis. Cells were pretreated with shikonin or left untreated as control for 24 h before incubating with or without gemcitabine for 48 h. (A) Cell viability assay. The cell viability index was determined by CCK-8 assay. (B) Isobologram plots for the interaction between shikonin and gemcitabine. Combination index (CI) versus fraction affected (Fa) plots obtained from median-effect analysis of Chou–Talalay. CI values: >1, antagonism; 1, additivity;
Contents lists available at ScienceDirect
Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm
Shikonin suppresses tumor growth and synergizes with gemcitabine in a pancreatic cancer xenograft model: Involvement of NF-kB signaling pathway Yongwei Wang a, Yinan Zhou a, Guang Jia a, Bing Han a, Ji Liu a, Yueqiu Teng b, Jiachen Lv a, Zengfu Song a, Yilong Li a, Liang Ji a, Shangha Pan a, Hongchi Jiang a, Bei Sun a,* a b
Department of Pancreatic and Biliary Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin 150001, China Department of Central Laboratory of Blood Cancer, The First Affiliated Hospital of Harbin Medical University, Harbin 150001, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 December 2013 Received in revised form 28 January 2014 Accepted 30 January 2014 Available online 9 February 2014
Although gemcitabine is currently the best chemotherapeutic agent available for the treatment of advanced pancreatic cancer, eventual failure of response is a significant clinical problem. Therefore, novel therapeutic approaches against this disease are highly needed. The aim of this study was to evaluate whether shikonin, a naphthoquinone derivative, has potential in the treatment of pancreatic cancer when used either alone or in combination with gemcitabine. Our in vitro results showed that shikonin inhibited the proliferation of three different human pancreatic cancer cell lines and potentiated the cytotoxic effect of gemcitabine, which correlated with the down-regulation of constitutive as well as gemcitabine-induced activation of NF-kB and NF-kB-regulated gene products. Most importantly, using a xenograft model of human pancreatic cancer, we found shikonin alone significantly suppressed tumor growth and argumented the antitumor activity of gemcitabine. These effects also correlated with the down-regulation of NF-kB activity and its target genes, decreased proliferation (PCNA and Ki-67), decreased microvessel density (CD31), and increased apoptosis (TUNEL) in tumor remnants. Collectively, our results suggest that shikonin can suppress the growth of human pancreatic tumors and potentiate the antitumor effects of gemcitabine through the suppression of NF-kB and NF-kB-regulated gene products. ß 2014 Elsevier Inc. All rights reserved.
Keywords: Shikonin Gemcitabine Pancreatic Cancer NF-kB
1. Introduction Pancreatic cancer remains one of the most fatal human malignancies and represents the fourth leading cause of cancer mortality in western countries, which has an alarmingly low 5year survival rate with best treatments available today of less than 5% [1,2]. The National Cancer Institute estimated that 45,220 men and women in the United States were diagnosed with pancreatic cancer in 2013 and 38,460 died of the disease [2]. Gemcitabine, erlotinib and abraxane are the only FDA-approved standard therapies for the treatment of patients with pancreatic cancer. However, they produce a poor response rate and are associated with various side effects and the development of chemoresistance, which exactly offsets the marginal benefit. Hence, the need to identify novel strategies against this fatal disease is imperative. Multiple lines of evidence suggest that the activation of master transcription factor NF-kB and NF-kB-regulated gene products * Corresponding author. E-mail address: [email protected] (B. Sun). http://dx.doi.org/10.1016/j.bcp.2014.01.041 0006-2952/ß 2014 Elsevier Inc. All rights reserved.
have a defined central role in growth, invasion, metastasis, angiogenesis, antiapoptosis and chemoresistance of pancreatic cancer [3–5]. First, NF-kB is constitutively active in pancreatic cancer and correlates with tumor progression and poor prognosis [6,7]. Second, it has the ability to promote pancreatic cancer growth partly via inhibition of apoptosis [8]. Third, NF-kB provides a direct stimulus toward cellular proliferation through the induction of growth promoting genes such as c-Myc and cyclin-D1, the latter of which has been shown to be overexpressed in human pancreatic cancer cell lines and cancer tissues and to have a significant inverse correlation with patient survival [9,10]. Fourth, NF-kB plays a pivotal role in the enhanced angiogenic potential of pancreatic cancer cells via increasing the production of proangiogenic factors such as VEGF [11]. Fifth, activation of NFkB is clearly associated with the migration and invasion of pancreatic cancer cells [12]. Finally, NF-kB contributes to the chemoresistance of pancreatic cancer cells [13]. Together, this evidence implicates the involvement of NF-kB pathway in pancreatic cancer and suggests that agents that can interfere with activation of NF-kB have enormous potential to suppress
Y. Wang et al. / Biochemical Pharmacology 88 (2014) 322–333
323
Fig. 1. Effect of shikonin on cell viability. (A) Chemical structure of shikonin. (B) Cell viability assay. Three pancreatic cancer cells (BxPC-3, PANC-1 and AsPC-1) and normal rat skeletal muscle cells (L6) were treated with varying concentrations of shikonin for 24, 48 and 72 h and cell viability was measured by CCK-8 assay. Data shown are representative of at least three independent experiments. * P < 0.05; ** P < 0.01 versus control.
tumor growth and may also serve to improve the effectiveness of gemcitabine. Shikonin (Fig. 1A), a natural naphthoquinone, is one such agent. Derived from the Chinese medical herb Lithospermum erythrorhizon, shikonin has been widely used as a traditional Chinese medicine for thousands of years to treat sore throats, macular eruptions, measles, carbuncles and burns [14,15]. Recently, emerging evidence suggests that shikonin has high efficacy against a series of human cancer cell lines in vitro and in several animal models in vivo with minimal or no toxicity to nonmalignant human cells [16–22]. The antitumor effect of shikonin may be associated with its ability to inhibit the expression of anti-apoptotic BCL-2 family members [23], activate caspases [24,25], decrease proteasome activity [22], suppress EGFR phosphorylation [26], generate reactive oxygen species (ROS) [27,28], arrest the cell cycle through p53 upregulation [29], activate the stress-related c-Jun-N-terminal kinase (JNK) pathway [30], and inactivate Akt and NF-kB pathways [21,31]. Collectively, these scientific findings suggest that shikonin may have a strong potential for future use in cancer prevention and treatment. However, the effect of shikonin on the growth of pancreatic cancer is not known. It is also not known whether shikonin has potential to act as a chemosensitizing agent to improve the therapeutic index of gemcitabine in pancreatic cancer cell lines and xenograft models.
In the present study, we investigated whether shikonin alone could suppress the growth of human pancreatic cancer cells both in vitro and in a xenograft mouse model, and whether shikonin could enhance chemosensitivity to gemcitabine. We found that shikonin inhibited the proliferation of various pancreatic cancer cells, potentiated gemcitabine-induced growth inhibition and apoptosis in pancreatic cancer cells in vitro and significantly enhanced the antitumor effects of gemcitabine in human xenograft pancreatic cancer model through the modulation of NF-kB and NF-kB-linked gene products.
2. Materials and methods 2.1. Materials Shikonin was purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Gemcitabine (Gemzar) was purchased from Eli Lilly. Antibodies used in this study included Antibodies against Survivin, procaspase-3, procaspase-9, c-Myc, Bcl-2, Bcl-xL, VEGF, PCNA and XIAP (Santa Cruz Biotechnology, CA, USA), Antibodies against Ki-67 and CD31 (Abcam Inc., MA, USA), and Antibodies against p-IkB-a, IkB-a, cyclin D1, COX-2, PARP (Cell Signaling Technology, Inc., MA, USA). Nuclear Extract Kit and Trans-AM NF-kB p65 ELISA Kit were obtained from Active Motif (Carlsbad, CA, USA).
324
Y. Wang et al. / Biochemical Pharmacology 88 (2014) 322–333
2.2. Treatment of cells Shikonin dissolved in DMSO [final concentration, 0.1% (v/v)] was used for the treatment of cells. The subconfluent cells (60–70% confluent) were treated with various concentrations of shikonin alone, gemcitabine alone or their combinations (as indicated in the figure legends) in complete cell culture medium and cells treated with vehicle (DMSO) served as control. The following experiments were repeated thrice. 2.3. Cell culture The pancreatic cancer cell lines PANC-1, BxPC-3 and AsPC-1 were obtained from the American Type Culture Collection (Manassas, VA). Rat normal skeletal muscle cell lines L6 were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China. PANC-1 and L-6 were cultured in DMEM and BxPC-3 and AsPC-1 was cultured in RPMI 1640. All media were supplemented with fetal bovine serum (10%), penicillin (100 U/ml) and streptomycin (100 mg/ml) (Irvine ScientiWc, Irvine, CA) and maintained at 37 8C in humidified air with 5% CO2 (all reagents were from HyClone China Ltd., China). 2.4. Cell viability assay The viability of treated cells was determined by using Cell Counting Kit-8 (CCK-8) kit (Dojindo Laboratories, Kumamoto, Japan) following the instructions outlined by the manufacturer and as previously described by us [32]. 2.5. Combination index analysis To evaluate the synergistic efficacies of shikonin and gemcitabine, the combination index (CI) isobologram method of Chou and Talalay was used. This commonly used analysis involves plotting concentration–effect curves for each agent and for multiply diluted fixed-ratio combinations by using the medianeffect equation and the combination index equation. A value of CI less than, equal to or greater than 1 indicates synergism, additivity and antagonism, respectively. The combination index values were calculated at multiple effect levels and the isobolograms plotted. 2.6. Apoptosis assay Apoptosis was evaluated by using (i) Cell Death Detection ELISA kit (Roche, Palo Alto, CA, USA) or (ii) Annexin V-FITC kit (BD Biosciences, San Jose, California, USA) following the instructions outlined by the manufacturer. 2.7. Caspase activity assay Caspase-3 and caspase-9 activities were measured using the Caspase Activity Kit (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China) following the instructions outlined by the manufacturer and as previously described by us [32]. 2.8. Western blot analysis To determine the levels of protein expression, cell lysates were prepared and subjected to Western blot analysis as described previously [32].
at 37 8C. The cells were then transfected with NF-kB p65 cDNA, control empty vector (pcDNA3.0), NF-kB p65 siRNA and control siRNA, respectively, using Lipofectamine 2000 (Invitrogen). The transfections were performed following the instructions outlined by the manufacturer. After transfection, cells were treated as designed before being harvested for the following studies. 2.10. Xenograft mouse model Animal studies were in accordance with the institutional guide for the care and use of laboratory animals. Our experimental protocol was reviewed and approved by the Animal Care and Use Committee of Harbin Medical University (Harbin, Heilongjiang, China). Male nude BALB/c mice (4–6 weeks old) were obtained from Shanghai Experimental Animal Center of the Chinese Academy of Sciences. Tumors were established by subcutaneous injection of 5 106 BxPC-3 cells into the flanks of mice. Tumor size was measured every three days with calipers and the volume was estimated according to the formula: V = (p/6) (larger diameter) (smaller diameter)2. When tumors reached around 100 mm3, the mice were randomly assigned to four groups (each group had eight mice): (a) control (DMSO was dissolved in 200 ml PBS, once daily by i.p. injection); (b) shikonin (2 mg/kg, once daily by i.p. injection); (c) gemcitabine (100 mg/kg, twice weekly by i.p. injection); and (d) shikonin and gemcitabine, following the same schedule of individual drugs. The doses of shikonin and gemcitabine selected for this experiment were based on preliminary experiments and previous studies. The mice were closely monitored for 24 days, then euthanized, and the tumors were removed. Each tumor was divided into two pieces: one fixed in 10% buffered formalin, and the other kept at 80 8C for further analysis. 2.11. PCNA and Ki-67 immunohistochemistry The methodology has been described previously [32]. In brief, paraffin-embedded tissue sections (5 mm) were immunostained with an anti-PCNA or anti-Ki-67 Ab. The number of PCNA or Ki-67 positive cells was counted in randomly selected ten microscopic fields at 400 magnification. 2.12. Quantification of apoptosis in tumor sections The methodology has been described previously [32]. In brief, paraffin-embedded tissue sections (5 mm) were stained with the TUNEL agent (Roche, Shanghai, China), and the number of TUNELpositive cells was counted in randomly selected 10 microscopic fields at 400 magnification. 2.13. Tumor microvessel density The methodology has been described previously [34]. In brief, tumor sections prepared from tumors 24 days after treatment were immunostained with anti-CD31 Ab. The number of microvessels was counted in randomly selected ten microscopic fields at 400 magnification, and the microvessel density was recorded. 2.14. Immunohistochemical analysis for cyclin D1, MMP-9, and VEGF in tumor samples The expression of cyclin D1, MMP-9, and VEGF were evaluated by immunohistochemical analysis as described previously [34]. 2.15. NF-kB DNA-binding activity assay
2.9. Plasmids and transfections The methodology has been described previously [33]. In brief, BxPC-3 cells were seeded in six-well plates and incubated for 24 h
The NF-kB DNA-binding activity was determined using the Trans-Am NF-kB/p65 ELISA Kit following the instructions outlined by the manufacturer and as described previously [35].
Y. Wang et al. / Biochemical Pharmacology 88 (2014) 322–333
2.16. Statistical analysis Data are represented as mean values standard deviation (SD). Statistical comparisons were made with a one-way analysis of variance (ANOVA) followed by Dunnett’s t test for multiple comparisons. A P value of less than 0.05 was considered significant.
3. Results The objective of this study was, first, to determine whether shikonin has potential in the treatment of pancreatic cancer when used either alone or in combination with gemcitabine and, second, to explore the molecular mechanism(s) behind these effects. Three different human pancreatic cancer cell lines (BxPC-3, PANC-1 and AsPC-1) of different origin were selected for this investigation. To monitor tumor growth in vivo, one of these cell lines, BxPC-3 was subcutaneously injected and used in a mouse xenograft model. 3.1. Effect of shikonin on cell viability To investigate the effect of shikonin on the proliferation of human pancreatic cancer cells, we used BxPC-3, PANC-1, and AsPC-1 cells, all of which exhibit differential resistance pattern to conventional chemotherapeutic drugs. The cells were assessed for viability using a CCK-8 assay following exposure to increasing concentrations of shikonin (0–10 mM) for 24, 48, or 72 h. As shown in Fig. 1B, shikonin suppressed the growth of all three human pancreatic cancer cells in a dose- and time-dependent manner. These data suggest that shikonin was an effective inhibitor of pancreatic cancer cell proliferation as a single agent. However, shikonin had minimal effect on normal rat skeletal muscle cells (Fig. 1B). 3.2. Shikonin potentiates growth inhibition caused by gemcitabine in pancreatic cancer cells We investigated the effects of shikonin and gemcitabine, alone or in combination, on cell viability by CCK-8 assay. For these studies, cells were pretreated with shikonin or left untreated as control for 24 h before incubating with or without gemcitabine for 48 h, and cell viability was determined at 72 h post-treatment. The doses of shikonin and gemcitabine used here were chosen based on our preliminary dose escalation studies and previous reports. As shown in Fig. 2A, treatment with either shikonin for 72 h or gemcitabine alone for 48 h resulted in only 25% to 40% growth inhibition in pancreatic cancer cells. However, pretreatment with shikonin for 24 h followed by treatment with gemcitabine for 48 h resulted in 65% to 80% growth inhibition in all three pancreatic cancer cell lines studied. The nature of the interaction between shikonin and gemcitabine was calculated by using the Chou/Talalay median-effect equation method for determining the combination index. A CI < 1 indicates synergy, and the CI range values for the combination of shikonin and gemcitabine in three different human pancreatic cancer cell lines were 0.4 to 0.9 for fractional effect corresponding to 0.3 to 0.9 (Fig. 2B), indicating that shikonin in combination with gemcitabine produces synergistic cell cytotoxicity in all three pancreatic cancer cells tested. These results show that shikonin in combination with low therapeutic doses of gemcitabine elicits significantly more loss of cell viability of pancreatic cancer cells than either agent alone, suggesting that therapeutic approaches devised for better cancer cell killing and fewer harmful side effects may be formulated. 3.3. Shikonin potentiates gemcitabine-induced apoptosis in human pancreatic cancer cells We next confirmed whether enhanced cytotoxicity by combination treatment of shikonin and gemcitabine was due
325
to induction of apoptosis. By histone DNA ELISA and Annexin V/ FITC flow cytometric analysis, we observed similar results showing that treatment with either shikonin or gemcitabine alone induced apoptosis in all three cell lines tested. Relative to single agents, shikonin pretreatment followed by gemcitabine treatment led to significantly higher apoptosis in the cancer cells (Fig. 2C and D). These results are consistent with cell viability assay, suggesting that greater cell growth inhibition resulting from combination treatment with shikonin and gemcitabine may due, at least partly, to the induction of more apoptosis in pancreatic cancer cells. 3.4. Molecular basis for shikonin and gemcitabine augmenting signal for apoptosis In an attempt to explore the detailed molecular mechanism(s) of shikonin-induced apoptosis and chemosensitization, we proceeded to assess the expression levels of procaspase-3, procaspase9 and cleavage of PARP, and modulation of the status of other apoptosis-related proteins. Western blotting revealed that both shikonin and gemcitabine alone down-regulated the expression of procaspase-3 and procaspase-9 in all three human pancreatic cancer cells and the combination was more effective (Fig. 2E). These findings were further confirmed by caspase activity assays showing that caspase-3 and caspase-9 activities were significantly increased by combination treatment with shikonin and gemcitabine (Fig. 2F). Since PARP is a substrate for caspase activity and a reliable marker of apoptosis, we next examined the level of cleaved PARP. Our data showed that whereas treatment of BxPC-3, PANC-1 and AsPC-1 cells with single agents showed low levels of PARP cleavage; shikonin pretreatment followed by gemcitabine treatment resulted in a stronger PARP cleavage (Fig. 2E). Furthermore, experiments were done to examine the expression levels of other apoptosis-related proteins. As shown in Fig. 3D, the level of c-Myc, COX-2, cyclin D1, Survivin, Bcl-2, Bcl-XL, XIAP, MMP-9 and VEGF were significantly decreased in the combination treatment group compared with control or single agent-treated group. Together, these results provide insight into the mechanism by which shikonin exerts its effects in pancreatic cancer cells. Since all of the above-mentioned proteins are known to be regulated by NF-kB, we examined whether shikonin exerts its effects by interfering with the NF-kB pathway. 3.5. Shikonin inhibits constitutive as well as gemcitabine-induced activation of NF-kB in pancreatic cancer cells Because NF-kB has been associated with both proliferation and apoptotic resistance, we next examined the effects of shikonin, gemcitabine, and the combination on NF-kB activity using an ELISA-based DNA-binding assay. Consistent with previous studies [32,34,36], gemcitabine alone was able to induce NF-kB DNA-binding activity (Fig. 3C). In addition, our results showed that NF-kB p65 siRNA transfection significantly inhibited gemcitabine-induced NF-kB DNA-binding activity whereas NF-kB p65 cDNA transfection enhanced activation of NF-kB DNA-binding activity stimulated by gemcitabine (Fig. 4A and B). As shown in Fig. 3A and B, we further found that treatment with shikonin caused a concentration-dependent decrease in NF-kB DNA-binding activity in all three pancreatic cancer cells, which was associated with a concentrationdependent decrease in phospho-IkB-a with a concomitant concentration-dependent increase in the cytosolic levels of the IkB-a protein. More importantly, shikonin was also found to significantly suppress gemcitabine- or p65 cDNA transfectioninduced activation of NF-kB DNA-binding activity (Fig. 3C and
326
Y. Wang et al. / Biochemical Pharmacology 88 (2014) 322–333
Fig. 2. Effect of shikonin and gemcitabine on cell viability and apoptosis. Cells were pretreated with shikonin or left untreated as control for 24 h before incubating with or without gemcitabine for 48 h. (A) Cell viability assay. The cell viability index was determined by CCK-8 assay. (B) Isobologram plots for the interaction between shikonin and gemcitabine. Combination index (CI) versus fraction affected (Fa) plots obtained from median-effect analysis of Chou–Talalay. CI values: >1, antagonism; 1, additivity;
