Structure-Activity Studies on Therapeutic Potential of Thymoquinone Analogs in Pancreatic Cancer
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- Banerjee, S., Azmi, A.S., Padhye, S. et al. Pharm Res (2010) 27: 1146. doi:10.1007/s11095-010-0145-3
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Pancreatic cancer (PC) is one of the deadliest of all tumors. Previously, we were the first to show that Thymoquinone (TQ) derived from black seed (Nigella sativa) oil has anti-tumor activity against PC. However, the concentration of TQ required was considered to be high to show this efficacy. Therefore, novel analogs of TQ with lower IC50 are highly desirable.
We have synthesized a series of 27 new analogs of TQ by modifications at the carbonyl sites or the benzenoid sites using single pot synthesis and tested their biological activity in PC cells.
Among these compounds, TQ-2G, TQ-4A1 and TQ-5A1 (patent pending) were found to be more potent than TQ in terms of inhibition of cell growth, induction of apoptosis and modulation of transcription factor-NF-κB. We also found that our novel analogs were able to sensitize gemcitabine and oxaliplatin-induced apoptosis in MiaPaCa-2 (gemcitabine resistant) PC cells, which was associated with down-regulation of Bcl-2, Bcl-xL, survivin, XIAP, COX-2 and the associated Prostaglandin E2.
From our results, we conclude that three of our novel TQ analogs warrant further investigation against PC, especially in combination with conventional chemotherapeutic agents.
KEY WORDSapoptosispancreatic cancerthymoquinonethymoquinone analogs
Pancreatic cancer (PC), the fourth leading cause of cancer death in the United States, accounts for an estimated 37,680 new cases and 34,290 deaths in 2009 (1). This dismal outcome in survival of patients diagnosed with PC is in part due to lack of early detection, the indolent nature of disease progression and perceived de novo and acquired chemo-resistance to conventional cytotoxic agents (2–5). Additionally, the aggressiveness of PC could in part be due to enrichment of cancer stem cells (CSCs) during therapy and because of the fact that CSCs are highly resistant to conventional therapeutics and thus may contribute to poor therapeutic outcome. Therefore, to improve the survival outcome of patients diagnosed with PC without undesirable side effects normally associated with current therapies, novel approaches must be devised for this deadly disease. To that end, we have previously shown the therapeutic and chemopreventive potential of the “natural dietary agent” Thymoquinone (TQ) derived from black seed (Nigella sativa) oil (6).
Nigella sativa is a spice that grows in the Mediterranean region and in Western Asian countries, including India, Pakistan and Afghanistan. In folklore medicine, the seed is reportedly associated with diverse therapeutic benefits in bronchial asthma, dysentery, headache, gastrointestinal problems, eczema, hypertension and obesity (7). The bioactive compound derived from Nigella sativa oil is TQ, which has been shown to exhibit anti-tumor activities, including anti-proliferative and pro-apoptotic effects on cell lines derived from breast, colon, ovary, larynx, lung, myeloblastic leukemia and osteosarcoma (8–13). It has also inhibited hormone refractory prostate cancer by targeting androgen receptor and transcription factor E2F (14). Mechanistically, as proposed by us and others, TQ reportedly induces apoptosis in tumor cells by suppressing NF-κB, Akt activation and extracellular signal-regulated kinase signaling pathways, and also inhibits tumor angiogenesis (6,15,16). Although a number of crucial genes are altered in PC, several important molecules stand out, including the COX family of proteins, which play critical roles in the initiation of cancer and acquisition of resistance to chemotherapeutics in human PC (17–19).
It is believed that PC arises from abnormal tissues or lesions in the pancreas known as pancreatic intraepithelial neoplasias (PanINs); by stalling the growth of PanINs, we hope to slow the development of, or prevent, PC. COX-2 is an enzyme that is up-regulated by growth factor and inflammatory cytokine-mediated activation of NF-κB in PC (20–23). Further studies on breast, colon, and PC have also shown that COX-2 plays a key role in aggressive tumor growth and metastases (24–29). Furthermore, we have confirmed that down-regulation of the “master transcription factor,” NF-κB, by natural agents such as TQ could lead to the sensitization of PC cells to cytotoxic conventional therapeutic agents, especially gemcitabine (6); however, we reasoned that the concentration used for this study may not be feasible for human patients. We therefore hypothesized that custom chemical synthesis of novel analogs of TQ with lower IC50 and with proven biological activity would be a step forward toward the development of novel therapeutic agents for the treatment of PC. Here, we present the results of our novel studies and document for the first time the synthesis of novel analogs of TQ with lower IC50 and with better biological activity. These compounds were initially evaluated for their ability to inhibit cell viability and induction of apoptosis at concentrations that are lower than the parental TQ in a panel of PC cell lines with different molecular signatures, and further tested their ability to sensitize gemcitabine-resistant PC cell line (MiaPaCa-2) to enhanced killing by gemcitabine. Moreover, we evaluated one of these promising analogs in a murine model for testing its toxicity to normal cells.
MATERIALS AND METHODS
Cell Culture and Reagents
The human PC cell lines-MiaPaCa-2, BxPC-3, AsPC-1and HPAC were obtained from American Type Culture Collection (Manassas, VA). Colo 357 and L3.6pl cells were obtained from Dr. Paul Chaio at MD Anderson Cancer Center (Houston, TX). The cell lines were maintained in continuous exponential growth in Dulbecco modified Eagle’s medium (DMEM, Life Technologies, Inc., Gaithesburg, MD) supplemented with 10% FBS, 100 units/ml penicillin and 10 mg/ml streptomycin in a humidified incubator containing 5% CO2 in air at 37°C.
Antibodies were obtained from the following commercial sources: anti-mouse Bcl-2 and Bcl-xL antibody from Abcam (Cambridge, MA), anti-PARP antibody from Biomol Research (Plymouth, PA), Caspase-3 antibody from Cell Signaling (Beverly, MA), anti-XIAP and anti-survivin from (RandD Systems) and anti-β-actin from Sigma Chemical Co. (St. Louis, MO). TQ (Sigma Chemicals, Ontario, Canada) and the investigated analogs were dissolved in DMSO to make 20 mM stock solution. Oxaliplatin and gemcitabine were obtained from the pharmacy of our institute.
Combinatorial Screening and Synthesis of TQ Analogs
Cell Viability Inhibition by TQ and TQ Analogs
Pancreatic cancer cells (3 × 103 cells/well) harboring different molecular signatures (MiaPaCa-2, BxPC-3, AsPC-1, Colo 357, L3.6pl and HPAC cells) were seeded in 96-well culture plates and replaced the next day with fresh medium containing either TQ (10 µM) or the investigational 11 analogs, diluted from stock solution. The effect of TQ and the analogs on cell viability was examined by MTT assay. The formazan formed by metabolically viable cells was dissolved in isopropanol, and the absorbance was measured at 595 nm using a plate reader (TECAN, Durham, NC), and the data was plotted as percent viable cell relative to control.
Quantification of Apoptosis
Two protocols were employed to confirm apoptosis following treatments with TQ or our analogs. The Cell Apoptosis ELISA Detection Kit (Roche, Palo Alto, CA), which quantifies the cytoplasmic histone-associated DNA fragmentation, was used according to manufacturer’s protocol. Additionally, Annexin V-FITC assay was used, and apoptotic cells were evaluated according to the manufacturer’s instructions (BD Biosciences, San Jose, CA).
Protein Extraction and Western Blot Analysis
MiaPaCa-2 cells were plated and allowed to attach for 36 h. TQ or the analogs exhibiting promising results (TQ-2G, TQ-4A1 and TQ-5A1) (10 µM) were directly added to cell cultures at the indicated concentrations and incubated for 48 h. Cell lysates were prepared by suspending the cells in RIPA lysis buffer and subjected to routine Western blot analysis as described earlier (6). Thirty to forty micrograms of total protein was separated on SDS-PAGE, electro-transferred and probed with specific antibodies.
Caspase Activity Assays
Caspase-3 activity was measured in MiaPaCa-2-treated cells by colorimetric assay according to the manufacturer’s protocol (R&D Systems, Minneapolis, MN). The cells were treated as described under Western blot analysis.
Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared from treated samples, and EMSA was performed by incubating 10 µg of nuclear extract with IRDye™–700-labeled NF-κB oligonucleotide as described earlier (6). The DNA-protein complex formed was visualized by Odyssey Infrared Imaging System using Odyssey Software Release 1.1. For loading control, 10 µg of nuclear protein from each sample was subjected to Western immunoblotting for retinoblastoma protein.
Determination of PGE2
BxPC-3 cells were seeded in 6-well plates and treated with TQ (0–10 µM) and the analogs (TQ-2G, TQ-4A1 and TQ-5A1; 10 µM) for 24 h in serum-free media. Conditioned medium was collected and analyzed to determine the levels of PGE2, using PGE2 high-sensitivity immunoassay kit (R&D Systems, Minneapolis, MN).).
COX Activity Assay
COX-1 and -2 activities were determined using the COX activity assay that utilizes the peroxidase component of cyclooxygenases. The peroxidase activity is typically assayed colorimetrically by monitoring the appearance of oxidized N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) at 590 nm. Briefly, 1 × 106 cells were seeded in the dark clear-bottom 96-well plate, allowed to grow for 24 h, and further assayed for COX-1 and -2 activity following manufacturer’s recommendations (Cayman Chemicals, Ann Arbor,MI).
DNA Cell Cycle Analysis
MiaPaCa-2 cells were treated with TQ or the analogs (TQ-2G, TQ-4A1 and TQ-5A1) for 72 h using equimolar concentrations (10 µM). After treatments, the cells were collected by trypsinization, washed with cold PBS and subsequently fixed in 450 µl of ice-cold ethanol by incubating them for 1 h at 4°C. The cells were then centrifuged at 500 × g for 5 min, and the pellet was washed twice with cold PBS, suspended in 500 µl of PBS, and incubated with RNase (20 µg/ml, final concentration) for 30 min at 37°C. The cells were then stained with propidium iodide (50 µg/ml final concentration) for 1 h, and analyzed by flow cytometry.
Chemosensitization by TQ and TQ Analogs
MiaPaCa-2 cells were plated and incubated with medium containing either TQ or the analogs (TQ-2G, TQ-4A1 and TQ-5A1) at 10 µM concentrations for 48 h and exposed to 0.5 μM gemcitabine or 6.0 µg/ml of oxaliplatin for an additional 36 h. The effect of pretreatment with TQ or the analogs on cell viability and apoptosis was examined by MTT assay and ELISA cell apoptosis detection kit as described earlier, and the absorbance was measured using a plate reader (TECAN, Durham, NC).
Initial Toxicity Testing in SCID Mice
All in vivo studies were conducted in accordance with Wayne State University approved animal care and ethics committee guidelines and procedures. TQ analogs were administered both intravenously and as oral gavages to determine the maximum tolerated dose (MTD).
Data are represented as mean ± SD for the absolute values or percent of controls as indicated in the vertical axis legend of figures. Statistical significance was determined by ANOVA test. Results of p < 0.05 was considered statistically significant.
Effect of TQ and TQ Analogs on Cell Viability and Apoptosis Induction
Effect of TQ and Analogs on Cell Cycle Progression
TQ Analogs Inhibit Anti-apoptotic Molecules in MiaPaCa-2 Cells
TQ Analogs Inhibit Activation of NF-κB in MiaPaCa-2 Cells
To investigate whether TQ and the analogs could abrogate constitutively expressed NF-κB in vitro in MiaPaCa-2, cells were treated with 10 µM dose of TQ and the analogs for 48 h, and subjected to gel shift assay (EMSA) for assessing the DNA binding activity of NF-κB. As shown in Fig. 4A, the analogs (TQ-2G, TQ-4A1 and TQ-5A1) resulted in down regulation of the DNA binding effect of NF-κB in MiaPaCa-2 cells, which again is consistent with down-regulation of the transcriptional target genes of NF-κB, such as Bcl-2 family of anti-apoptotic proteins, survivin and XIAP.
Suppression of COX Activity and Prostaglandin (PGE2) Synthesis in BxPC3 Cells
In our previous study, we showed using Western immunoblotting that TQ inhibits COX-2 protein expression in HPAC pancreatic cancer cells (6). We confirmed the molecular interaction between the parent TQ and cycloxygenase enzyme by molecular docking studies (personal observation, non-communicated). Based on preliminary computer modeling data acquired, it was concluded that TQ binds into the active site of COX-2 with binding energy of −7.68 Kcl/mole, which indicates that the compound might be a good candidate for COX-2 inhibition (personal observation, non-communicated).Thus, building from our preliminary molecular docking studies, we anticipated that the analogs will show more potent docking to COX-2 and subsequently inhibit COX-2; therefore, we carried further studies related to these molecules of interest in PC cells with high basal expression of these enzymes.
TQ Analogs Sensitize PC Cells to Chemotherapeutic Agents
TQ Analogs Sensitize MiaPaCa-2 Cells to Apoptosis by Gemcitabine and Oxaliplatin
TQ and Its Analogs Are Non-toxic to Animals
Previously, we were able to administer TQ at 3 mg/mouse/day doses for 21 days without any signs of apparent toxicity to mice (6). Hence, we further evaluated the effect of one TQ analog (TQ-2G) on animal body weight and overall well being (data not shown). Based on empirical criterion, no evidence of any severe toxicity as inferred from body-weight loss criteria or signs of aversion to food intake or diarrhea were evident during the window of treatment. Furthermore, no macroscopic evidence of necrosis or hemorrhage in any visceral organs was seen under our experimental condition. It was inferred that TQ-2G, when tested in SCID mice at doses up to 50 mg/kg i.v. and up to 700 mg/kg by oral gavage, is non-toxic to mice, suggesting that this analog would be a non-toxic agent to normal cells. It also suggests the possibility of significant tumor-specific activity either alone or in combination with conventional therapeutic agents for the treatment of pancreatic tumors. However, further in-depth investigations are warranted for its safety.
Pancreatic cancer (PC) is one of the deadliest of the solid malignancies, with a five-year survival rate of only 4% (1). Growth inhibition and induction of apoptosis constitute the major mechanisms of action of most chemotherapeutics during cancer. Unfortunately, PC is inherently resistant to apoptosis in all conventional cancer therapeutic agents, which poses a great challenge to clinicians for the treatment of patients diagnosed with PC. Gemcitabine, oxaliplatin and 5-Flurouracil are the only approved drugs that have very modest activity against this disease. Studies from our laboratory and others have acknowledged that the underlying resistance of PC to these chemotherapeutic drugs could in part be due to the over-expression of chemo-resistant molecules such as NF-κB, Bcl-xL, survivin and COX-2. In recent years, increased attention has been directed towards naturally occurring compounds, particularly “dietary agents,” for their use as inhibitors of tumor progression and/or treatment due to their systemic, non-toxic nature and pleiotrophic molecular effects, which is believed to contribute to reversing drug resistance (30–33).
We recently reported the anti cancer properties of Thymoquinone (TQ), a natural non-toxic and bioactive compound derived from black seed (Nigella sativa) oil against PC (6,7). It has been proposed that TQ induces apoptosis in tumor cells by several mechanisms including NF-κB suppression, Akt activation and extracellular signal-regulated kinase signaling pathways, and also by inhibiting tumor angiogenesis (6,7,20,21). These studies clearly suggest that TQ could be useful as an adjunct to conventional chemotherapeutics, such as gemcitabine and oxaliplatin. Our data indicate that TQ alone at 25 µM was able to effectively induce cell growth inhibition and apoptosis in PC cells mediated through a mechanism involving inactivation of COX-2 and NF-κB (6). However, the concentration needed for such activity was viewed as practically high to be pharmacologically bioavailable for human clinical intervention studies, and thus we reasoned that novel synthetic analogs of TQ with proven bioactivity and lower IC50 than the parental TQ without systemic toxicity would be clinically relevant from translational viewpoint. This issue is of critical importance and highly relevant to cancer therapy, especially because any agent with low or no systemic normal tissue toxicity while retaining a robust anti-tumor activity is highly desirable.
To that end, we modified the original TQ to produce a series of 27 analogs, of which 11 are reported in this study. These modifications have been directed at the carbonyl sites or the benzenoid sites using single-pot synthesis that resulted in enhanced lipophilicity (for example the LogP for TQ-2G is 5.60 compared to 2.84 for TQ, and higher binding affinity to COX-2). Among these analogs investigated, three analogs, TQ-2G, TQ-4A1 and TQ-5A1 (patent pending), were found to be more potent than TQ in terms of their ability to inhibit cell growth and induce apoptosis in PC cells, and this superior biological activity of these novel analogs was found to be mechanistically linked to the down-regulation of transcription factor NF-κB and anti-apoptotic and cell survival-related molecules such as Bcl-2, Bcl-xL, survivin and XIAP. Additionally, our data provided new evidence in support of the activity of these analogs in inhibiting the production and secretion of PGE2 in high COX-2-expressing PC cells (BxPC-3). These selective analogs, TQ-2G, TQ-4A1 and TQ-5A1, have a much lower IC50 (<10 µM) compared to the parent TQ (IC50 ∼25 µM), which is more favorable and acceptable for clinical therapy.
In our previous study, TQ was shown to exhibit chemosensitizing effects to gemcitabine and oxaliplatin (6). This effect was attributed to the inactivation of DNA binding activity of NF-κB, resulting in the inactivation of multiple NF-κB downstream survival factors. In this study, we found a similar chemosensitizing effect by TQ analogs. TQ-2G, TQ-4A1 and TQ-5A1 were more potent than the parental TQ against PC in the sensitization of PC cells to gemcitabine or oxaliplatin treatment. These analoges exhibited strong potential for the down-regulation of anti-apoptotic molecules, such as Bcl-2, Bcl-xL, survivin and XIAP, compared to parental TQ, which may represent the molecular mechanism by which these compounds elicit their biological activity; however, further in-depth molecular and animal experiments are needed for confirming the superiority of these novel analogs as anti-tumor agents compared to the parental TQ. It is exciting to see that these analogs showed increased caspase-3 activity, which was associated with increased apoptosis of PC cells. Furthermore, our observations with the cell cycle progression revealed that these analogs possess differential effects on cell cycle progression with the analog TQ-5A1 showing strong G2/M cell cycle arrest, which suggests that this analog could be useful for combination therapy using radiation, which of course requires further in-depth investigations. A broad spectrum of cell cycle genes trigger the disruption of the cell membrane integrity in response to stress condition elicited by cell cycle arrest. This can be viewed as one speculative reason for the differential trend between cell viability and cell cycle progression, as noted with the analogs in the present study.
Although complete understanding of the underlying apoptosis-inducing mechanism of these novel analogs is required, the initial findings reported here are encouraging, especially the chemosensitizing effects of these analogs to conventional agents that are not very effective in the treatment of PC. Our findings on chemoresistant MiaPaCa-2 cells clearly suggest that these compounds may hold promise for combination therapy toward better treatment outcome of patients diagnosed with PC. Interestingly, our preliminary data suggest that TQ-2G is non-toxic in SCID mice at doses up to 50 mg/kg when administered through i.v. route and a dose up to 700 mg/kg when administered orally by gavage, which suggests that this analog could indeed be useful in combination therapy without added toxicity when combined with conventional therapeutics such as gemcitabine or oxaliplatin. Of added interest, the synthesized analogs described closely mirror Lipinski’s Rule of Five.
In conclusion, we presented preliminary evidence for novel synthesis of TQ analogs with biological activity that is better than the parental TQ without systemic toxicity; thus, we believe that these and other novel analogs of TQ, could serve as promising agents for better treatment outcome of patients diagnosed with pancreatic cancer.
The authors express their sincere appreciation to Ms.Christine Wojewoda for her editorial assistance. Grant support from the National Institutes of Health RO1CA109389 (RM Mohammad) and NIH R01CA083695, R01CA131151, and R01CA132794 awarded to FHS is gratefully acknowledged. The authors also acknowledge the financial contribution of Guido Foundation.