Zyflamend induces apoptosis in pancreatic cancer cells via modulation of the JNK pathway

Current pharmacological therapies and treatments targeting pancreatic neuroendocrine tumors (PNETs) have proven ineffective, far too often. Therefore, there is an urgent need for alternative therapeutic approaches. Zyflamend, a combination of anti-inflammatory herbal extracts, that has proven to be effective in various in vitro and in vivo cancer platforms, shows promise. However, its effects on pancreatic cancer, in particular, remain largely unexplored. In the current study, we investigated the effects of Zyflamend on the survival of beta-TC-6 pancreatic insulinoma cells (β-TC6) and conducted a detailed analysis of the underlying molecular mechanisms. Herein, we demonstrate that Zyflamend treatment decreased cell proliferation in a dose-dependent manner, concomitant with increased apoptotic cell death and cell cycle arrest at the G2/M phase. At the molecular level, treatment with Zyflamend led to the induction of ER stress, autophagy, and the activation of c-Jun N-terminal kinase (JNK) pathway. Notably, pharmacological inhibition of JNK abrogated the pro-apoptotic effects of Zyflamend. Furthermore, Zyflamend exacerbated the effects of streptozotocin and adriamycin-induced ER stress, autophagy, and apoptosis. The current study identifies Zyflamend as a potential novel adjuvant in the treatment of pancreatic cancer via modulation of the JNK pathway. 5rXAVcWGwP9WqSeTdiEBY7 Video abstract Video abstract

that in 2019, pancreatic cancer will be the third leading cause of cancer-related death [1]. Although advancements in science and health care have led to decreased mortality from numerous forms of cancer, pancreatic cancer survival rates have not improved significantly over the past several decades, leaving a desperate need for more effective treatment options. The risk of developing pancreatic cancer has been associated with numerous biological, environmental, pathological, and genetic factors. These factors include variables such as familial history, chronic pancreatitis, smoking, obesity, and diabetes (reviewed in [2,3]). In addition, hereditary familial factors and germline mutations could contribute to increased risk of cancer onset. However, the survival outcome and treatment effectiveness are highly dependent upon the time of diagnosis. While pancreatic ductal adenocarcinoma (PDAC) is the most commonly contracted and investigated subtype, the pathological nature of the rarer pancreatic neuroendocrine tumors (PNETSs) remains elusive [4].
PNETs account for less than 5% of all pancreatic cancers and are often diagnosed at a late stage in patients with advanced metastasis making surgery a non-viable treatment option [5,6]. Additionally, because of their heterogeneous clinical presentation and responses to chemotherapeutic agents, current pharmacological therapies and treatment options targeting PNETs have too often proven in-effective [4]. PNETs treatment options often include the use of chemotherapeutic compounds such as streptozocin, 5-fluorouracil, doxorubicin, and cisplatin, both alone or in combination (reviewed in [6,7]). The effectiveness of these compounds often increases at higher doses, but this directly exacerbates the risk for cytotoxicity and collateral side effects [8]. In addition, adjunct therapy involving the combination of various treatment approaches, such as surgery and radiotherapy, is often implemented [9]. In pursuit of survival and improved quality of life, patients often seek to enhance the effectiveness of conventional therapies through dietary and supplemental means [10,11].
New Chapter (Brattleboro, VT) first launched Zyflamend based on the idea of combining extracts of ten different herbs to effectively reduce inflammation through cyclooxygenase (COX) inhibition [12]. A large volume of research has emerged over the last two decades that supports the anti-inflammatory properties of Zyflamend and its ability to inhibit COX in various types of cancer, including prostate [13], melanoma [14], and oral cancer [15]. Individually, many of the extracted components of Zyflamend have proven to exhibit anti-cancer activity [16,17]. However, the high doses required to optimize effectiveness against cancer could prove infeasible for the majority. In theory, the combined effects generated through integrating these unique and powerful herbs could grant superior benefit over their isolated form [18]. Additionally, Zyflamend has shown the capability to interact with a variety of integral cellular signaling pathways beyond COX. These signaling pathways and mechanisms of interaction include: AMP-activated protein kinase (AMPK) [19,20], nuclear factor kappa-lightchain-enhancer of activated B cells (NF-κB) [21,22], mammalian target of rapamycin (mTORC1) [20], apoptosis, cell growth [13,23], endoplasmic reticulum (ER) stress [22,24,25] and finally autophagy [14]. While these studies show that Zyflamend could exhibit profound potential in the therapeutic application, more research is required to elucidate the molecular basis underlying its anti-cancer effects. In the current study, we investigated the effects of Zyflamend on the survival of beta-TC-6 pancreatic insulinoma cells (β-TC6) and deciphered the underlying molecular mechanisms.

Zyflamend treatment
Zyflamend was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 800 mg/ml. Cells were treated with Zyflamend at the indicated concentrations and for the indicated durations. Treatments were terminated by two washes with ice-cold phosphate buffer saline (PBS). Plates were then flash-frozen in liquid nitrogen and stored at − 80°C until further analyses.

Proliferation assay
Cell proliferation assay was performed using the sulforhodamine B (SRB; Millipore-Sigma) method as previously described [27] with modification. Briefly, an equal number of β-TC6 cells (1 X 10 6 cells) were seeded in 6 well plates. Six h. later cells were treated with the indicated concentrations of Zyflamend and incubated at 37°C in an atmosphere of 10% CO 2 for the indicated time. Treatment was stopped by two washes with ice cold PBS and cells were fixed with 17% trichloroacetic acid in PBS. Intracellular proteins were stained for 10 min at room temperature using 0.4% SRB dissolved in 1% acetic acid. Excess SRB stain was removed by rinsing the plates thoroughly with running tap water. Plates were air-dried for at least 6 h prior to dissolving the stain in 10 mM Tris (pH 9.0). Intracellular proteins were quantified using the Synergy™ HTX Multi-Mode microplate reader (BioTek Instruments, Inc. Winooski, VT) at a wavelength of 540 nm. The relative survival rates of cells were determined by dividing the absorbance observed for a given treatment by the absorbance detected in control cells treated with DMSO, and expressed as a fold change.

Colognic test
Colonie formation assay was performed as previously described [29,30] with modification. Cells were seeded in the presence of DMSO (control), Zyflamend alone or in combination with Z-VAD.fmk (10 uM), 4-PBA (250 uM), SP600125 (10 uM), STZ (5 mM) or adriamycin (5 μM). After 24 h, media was replaced with a freshly prepared new cell culture media and plates were incubated for 7 days at 37°C in an atmosphere of 10% CO 2 . After incubation, the colonies were washed with ice-cold PBS, fixed and stained with a mixture of 6.0% glutaraldehyde and 0.5% crystal violet for 30 min. The plates were washed with water, dried, and colonies (with more than 30 cells/colony) were counted. The relative number of colonies in each condition was determined by dividing the number of colonies for a given treatment by the total number of colonies in DMSO treated cells (control), and expressed as a percentage relative to DMSO-treated cells (Ctrl.).

Western blotting analysis
Cells were lysed in radio-immunoprecipitation assay (RIPA) buffer, as previously described [31]. Lysates were clarified by centrifugation at 15,000 g for 10 min, and protein concentrations was determined using bicinchoninic acid assay kit (Pierce Chemical, Dallas, TX). Proteins (5-30 μg) were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Immunoblotting of lysates was performed with primary antibodies (Supplementary Table 1), and after incubation with secondary antibodies, proteins were visualized using Luminata™ Forte Western Chemiluminescent HRP Substrate (Millipore-Sigma). Pixel intensities of immunoreactive bands were quantified using FluorChem Q Imaging software (Alpha Innotech Corp., San Leandro, CA). Data for phosphorylated proteins are presented as the intensity of phosphorylation normalized to total protein expression, while total protein expression was normalized to the loading control: β-actin.

Morphological analysis of apoptosis
β-TC6 cells were exposed to Zyflamend for the indicated duration then washed with PBS and labeled with Hoechst 33258 (50 μg/ml in PBS) (blue-green fluorescence). Hoechst binds to condensed nuclear chromatin [31] and was used to visualize apoptotic cells (green fluorescence) by fluorescence microscopy (Leica DMI8, Leica Microsystems Inc. Buffalo Grove, IL). For each condition, at least 500 cells were counted. Percentages of apoptotic cells were calculated relative to total cells.

Annexin V staining
Quantification of externalized phosphatidylserine, an early event in the apoptotic cascade, was performed using flow cytometry as previously described [32] with modification. Briefly, 50-60% confluent β-TC6 cells were exposed to Zyflamend for 24 h then washed with PBS and resuspended in 200 μl of PBS containing 10% FBS. Immediately after, an equal volume of the 2X Guava Nexin reagent (Millipore-Sigma) containing Annexin V-Fluorescein isothiocyanate (FITC) and 7amino-actinomycin D (7-AAD) was added to each treatment and incubated for 10 min at room temperature under light-protected conditions. Intensities of fluorescence emitted by Annexin V-FITC and 7-AAD were measured using the Guava® easyCyte Flow Cytometer (Millipore-Sigma) on PM1 and PM2 channels, respectively. Viable (negative for both Annexin V and 7-AAD staining) and apoptotic cells, both at early (Annexin V positive, 7-AAD negative) and late (positive for both Annexin V and 7AAD) stages were quantified using the InCyte™ and GuavaSuite Software package (Luminex Corp; Austin, TX).

Cell cycle analysis
Cell cycle analysis was conducted through assessing the DNA content of cells stained with propidium iodide as previously described [33] with modification. Briefly, 50-60% confluent β-TC6 cells were starved in 0.1% serum media for 16 h, then complete growth media was added to the cells along with various freshly prepared concentrations of Zyflamend. 24 h later, cells were harvested, washed twice with ice-cold with PBS, and fixed overnight in 70% ethanol at 4°C. Next, cells were washed twice with ice-cold PBS and incubated in a freshly prepared RNase solution [10 mM Tris-HCl, pH 7.4 containing 100 U/ml of DNase-free RNase A (Applied Biosystems, Austin, TX)] for 30 min at 37°C. Cells were washed twice with ice-cold PBS and incubated in a solution of propidium iodide (PI; 10 μg/ml in PBS) overnight at 4°C under light protected conditions. Fluorescence intensity of PI was measured using the Guava® easyCyte flow cytometer on PM2 channel. DNA histogram analysis was performed on 5000 cells using the InCyte™ and GuavaSuite Software package and the proportions of cells with one or two copies of their chromosomal DNA were calculated.

Statistical analysis
Data were analyzed using JMP Pro 14.2 program (SAS Institute, NC) and presented as means + standard error of the mean (SEM). Unpaired heteroscedastic two-tail Student's t test was used for all statistical analyses, and differences were considered significant at p < 0.05. Single symbol (such as * or †) was used to indicate a p value that is less than 0.05, while double symbol (such as ** or † †) corresponds to a p value that is less than 0.01.

Results
Zyflamend decreases cell proliferation, causes G2/M cellcycle arrest, and induces apoptotic cell death in pancreatic cancer cells We first examined the effects of varying doses of Zyflamend on the proliferation of pancreatic insulinoma β-TC6 cells. Zyflamend caused a significant dose-and time-dependent decrease in cell growth (Fig. 1a). Additionally, a Zyflamend dose of 25 μg/ml was sufficient to inhibit cell proliferation by 58% after 36 h of treatment, while a dose of 800 μg/ml completely abolished cell proliferation (Fig. 1a). In line with these findings, cell cycle analysis demonstrated that Zyflamend alters cell cycle distribution in a dose-dependent manner. Indeed, Zyflamend treatment resulted in the enrichment of the G2/M fraction with 2 N DNA content, which was accompanied by a reduction in cell cycle progression through the G0/ G1 and S phases ( Fig. 1b-c). These results suggest that Zyflamend-induced inhibition of cell proliferation is mediated, at least in part, through cell cycle arrest in the G2/M phase.
In order to determine whether Zyflamend-induced inhibition of cell proliferation was associated with apoptotic cell death, we determined changes in apoptosis in β-TC6 cells treated with increasing doses of Zyflamend (0, 25, 50, 100, 200, 400, and 500 μg/ml) for 24 h using two approaches: the Guava Nexin Annexin V assay and Hoechst 33258 stain. Using the Annexin V assay, the percentages of both Annexin V positive/7-AAD negative cells (reflective of early apoptotic cells), and Annexin V positive/7-AAD positive cells (reflective of late apoptosis), exhibited a dose-dependent and significant increase in response to Zyflamend treatment ( Fig. 1d-e). Consistent, with this observation, the number of Hoechst-positive cells was also higher in Zyflamendtreated cells compared to control cells ( Fig. 1f-g). Hoechst 33258 is a nucleic acid dye that binds to condensed chromatin in the nucleus of apoptotic cells, thus giving an assessment of overall apoptotic cell death [34]. At a dose of 200, 400 and 500 μg/ml, the percentages of apoptotic cells were 30.44 ± 4.07%, 40.30 ± 1.30%, and 53.75 ± 4.45%, respectively, further emphasizing the proapoptotic effects of Zyflamend on these cells. Similar findings were obtained using the MTT assay (Fig. 1h).

A human equivalent dose of Zyflamend induces apoptotic cell death in β-TC6 cells
To further characterize the pro-apoptotic properties of Zyflamend, we conducted a time course analysis using a physiological relevant fixed dose of Zyflamend (200 μg/ ml) [18]. This dose is representative of the maximum plasma concentration of a primary ingredient of Zyflamend (curcumin) that was reported in humans after oral administration [18]. At this dose, a marked increase in chromatin condensation and apoptotic cell number was observed after 24 h of treatment ( Fig. 2a-b). Subsequently, markers of apoptosis and cell survival were investigated using Western blotting. Zyflamend induced cleavage of caspase-3 and its downstream target poly (ADP-ribose) polymerase (PARP) (Fig. 2c-d). In addition, we examined changes in the mitogen-activated protein kinases (MAP kinases) pathways in response to Zyflamend. Our data revealed that β-TC6 cells treated with Zyflamend exhibited a marked decrease in the phosphorylation of protein kinase B (AKT) and extracellular signal-regulated kinases (ERK), particularly after 12 h of treatment.
To determine whether Zyflamend-induced cell death was associated with the caspase dependent pathways of apoptosis, we tested whether blocking caspases' activity using carbobenzoxy-valyl-alanyl-aspartyl-[Omethyl]-fluoromethylketone (Z-VAD.fmk) could inhibit Zyflamend-induced chromatin condensation and apoptosis. Z-VAD.fmk is a potent cell permeable pan-caspase inhibitor, which acts by irreversibly binding to the catalytic site of the caspase proteases, and thus inhibiting their activities. Our study shows that pretreatment with Z-VAD.fmk caused a significant decreased in the levels of chromatin condensation in Zyflamend-treated cells ( Fig. 2e-f). Additionally, Z-VAD.fmk treatment alleviated Zyflamend-induced cell toxicity as judged by the MTT (Fig. 2g) and the colony formation ( Fig. 2h-i) assays. Taken together, our findings indicate that Zyflamend treatment reduces cell viability and induces cell death through the induction of the apoptotic machinery in β-TC6 cells.

Zyflamend induces ER stress, apoptosis and autophagy responses in β-TC6 cells
A plethora of intrinsic and extrinsic pathways can lead to apoptosis in response to stressors, including ER stress, and autophagy, among many more. Therefore, in order to dissect the precise molecular mechanism mediating the pro-apoptotic effects of Zyflamend, we examined the activation of key signaling molecules related to these pathways. Zyflamend (200 μg/ml) significantly induced ER stress as judged by the activation of ER  transmembrane sensors, protein kinase RNA-like endoplasmic reticulum kinase (PERK) and inositol-requiring transmembrane kinase/endoribonuclease 1α (IRE1α), along with downstream targets such as eukaryotic translation initiation factor 2 alpha (EIF2α) and C/EBP homologous protein (CHOP) by Western blotting. Zyflamend induced ER stress as evidenced by increased PERK (Thr 980 ), EIF2α (Ser 51 ) and IRE1α (Ser 724 ) phosphorylation (Fig. 3a). Furthermore, the level of CHOP expression was elevated, a direct downstream target of both the PERK and IRE1 pathways. The activation of CHOP, a potent inducer of apoptotic cell death [35,36], in response to ER stress ( Fig. 3a-b), strengthens our conclusions of Zyflamend-induced apoptosis in these cells. Moreover, Zyflamend has been shown to activate AMPK, and our results recapitulate these previous findings [20]. The AMPK signaling pathway has been shown to regulate autophagy and cell death [37]. Therefore, in order to assess whether Zyflamend induces autophagy in β-TC6 cells, we immunoblotted for autophagy-related proteins. We observed a time dependent increase in beclin 1, microtubule-associated proteins 1A/1B light chain (LC3-I & II), and autophagy-related proteins 5 and 7 (ATG5/7) (Fig. 3c-d). The increase in the expression of these proteins is indicative of elevated autophagy in these cells. Because ER stress, inflammation, and autophagy can all lead to apoptosis, we used the pan-caspase inhibitor Z-VAD.fmk to determine which pathway might be responsible for the pro-apoptotic effects of Zyflamend. Our data shows that, while there was a significant attenuation of Zyflamend-induced cleavage of caspase-3 and its downstream target PARP, treatment with Z-VAD.fmk had no effects on Zyflamend-induced activation of the AMPK, autophagy, and ER stress signaling cascades (Fig. 3e-f). These findings suggest that ER stress, autophagy, and MAP kinases pathways are upstream of the apoptotic signaling cascade that might be mediating the pro-apoptotic effects of Zyflamend in β-TC6 cells.

Zyflamend-induced cell death is mediated through the ER stress/JNK/autophagy pathway
The exact molecular mechanism(s) leading to apoptosis by Zyflamend in cancer cells has/have not been revealed yet, although recent studies have supported the role of AMPK in the regulation of cancer cell growth, bioenergetics, autophagy, and cell death. To investigate the potential role of AMPK in Zyflamend-induced apoptosis, we pre-treated cells with the AMPK inhibitor compound C (CC; 5 μM) for 12 h prior to Zyflamend treatment for an additional 24 h. The dose and duration of exposure were determined based on the ability of compound C to reverse AMPK-dependent inhibitory phosphorylation of acetyl-CoA carboxylase (ACC) (data not shown). Cells were then examined for AMPK activation as well as activation of inflammation, ER stress, autophagy, and cell death (Fig. 4a). While the level of phosphorylated AMPK was reduced in co-treated cells, pre-treatment with compound C had no effects on Zyflamend-induced JNK phosphorylation, ER stress, autophagy, or cell death ( Fig.  4a-b). These data suggest that Zyflamend-induced apoptosis in β-TC6 cells is independent of AMPK activation. Next, we sought to examine whether blocking autophagy using 3-MA could protect cells against Zyflamendinduced apoptosis. 3-MA inhibits autophagy by blocking autophagosome formation via the inhibition of class I and class III phosphatidylinositol 3-kinases (PI3K) [38]. Cells were pre-incubated with 3-MA (5 nM) for 12 h prior to Zyflamend treatment. Co-treatment with Zyflamend and 3-MA significantly decreased the expression of beclin 1, LC3, and cleaved caspase-3, but had no effects on ER stress markers nor on AMPK phosphorylation (Fig. 4c-d). These data suggest that autophagy mediates Zyflamend-induced apoptosis and that both AMPK and ER stress activation by Zyflamend occur upstream of autophagy and apoptosis.
The relationship between these two fundamental processes, ER stress and autophagy, is complex and poorly understood. Recent literature demonstrates that both pathways display dual roles in cell survival in multiple  cancer cell lines. Similar to ER stress, autophagy has been shown to promote cell survival by clearing unwanted components from the cells. Nonetheless, a considerable body of evidence also indicates that both autophagy and ER stress can lead to apoptosis in tumor cells. In addition to this, a growing body of literature supports existing crosstalk between the two pathways [39,40]. However, which pathway is upstream of the other is yet to be determined. Our data suggest that Zyflamend-induced autophagy is likely to be downstream of ER stress. To test this hypothesis, we pretreated β-TC6 cells with an ER stress inhibitor 4phenylbutyrate (4-PBA; 5 mM for 12 h) prior to Zyflamend treatment, and we examined changes in inflammation, ER stress, autophagy, and cell death (Fig. 4e-f). 4-PBA is a cell permeant chemical chaperone that has been shown to inhibit ER stress and ER stress-induced apoptosis in many cancer cell types, including pancreatic cancer cells [41,42]. Our findings show a profound decrease in ER stress, autophagy, and cell death markers in response to Zyflamend (Fig. 4e-f) when cells were pretreated with 4-PBA. Additionally, 4-PBA treatment alleviated the decrease in cell proliferation (Fig. 4g) and colony formation (Fig. 4h-i) caused by Zyflamend. Furthermore, pre-treatment of β-TC6 cells with 4-PBA reduced Zyflamend-induced chromatin condensation ( Fig. 4j-k). Conversely, 4-PBA did not alter the activation of AMPK by Zyflamend (Fig. 4e-f), suggesting that ER stress occurs upstream of autophagy and apoptotic cell death.
Previous studies have shown that the ER stress sensor IRE1 may promote autophagy through the TRAF2/ ASK1/JNK pathway [43,44]. To test this hypothesis, we treated β-TC6 cells with SP600125 (a selective JNK inhibitor) and investigated changes in inflammation, ER stress and proliferation in response to Zyflamend treatment (Fig. 5). As expected, cells pre-treated with SP600125 exhibited a significant reduction in the phosphorylation of JNK and reduced expression of autophagy and cell death markers in response to Zyflamend (Fig.  5a-b). Likewise, JNK inhibition protected β-TC6 cells from Zyflamend-induced reduction in cell survival (Fig.  5c), colony formation (Fig. 5d-e), and chromatin condensation ( Fig. 5f-g). However, pre-treatment with SP600125 did not alter Zyflamend-induced activation of the AMPK and ER stress pathways ( Fig. 5a-b). Altogether, the above findings suggest that Zyflamend is capable of reducing survival and inducing apoptosis in the β-TC6 murine insulinoma cells through a complex mechanism involving ER stress-mediated JNK activation, which in turn induces autophagy followed by the induction of apoptosis.

Zyflamend enhances the effects of streptozotocin and adriamycin
It was our objective to assess the effectiveness of combining Zyflamend with chemotherapeutics commonly used to treat endocrine pancreatic cancer. Thus, we examined the effects of Zyflamend treatment alone or in combination with streptozotocin (STZ) or adriamycin (ADR) for 24 h. Total cell lysates were then immunoblotted for various markers of ER stress, autophagy, and cell death. Both STZ and ADR caused a significant increase in ER stress as demonstrated by the increased phosphorylation of both PERK and its downstream target EIF2α, along with increased phosphorylation of IRE1 and the splicing of its downstream target X-Box Binding Protein 1 (XBP1). Likewise, the expression of CHOP was significantly higher in cells treated with Zyflamend, streptozotocin, or adriamycin. Zyflamend treatment exacerbated the effects of streptozotocin and adriamycin on ER stress (Fig. 6a-b). Additionally, Zyflamend enhanced STZ and ADR-induced expression of autophagy (beclin 1, LC3-I & II, ATG5, and ATG7) and apoptosis (cleaved caspase-3) markers (Fig. 6c-d). Consistent with these findings, Zyflamend exacerbated STZ and ADRinduced cell toxicity as demonstrated by the colony formation (Fig. 6e-f) and the MTT (Fig. 6g) assays. Notably, these findings were corroborated in rat pancreatic insulinoma RIN-5F cells with a combination of Zyflamend and STZ or ADR (Supplementary Fig. S1). Taken together, our study revealed that Zyflamend may serve as (See figure on previous page.) Fig. 3 Zyflamend Induces Inflammatory, ER Stress, and Autophagy Responses in β-TC6 Cells. a-b Total cell lysates from control and Zyflamend treated cells for 3, 6, 12, 24, 36 and 48 h were immunoblotted for ER stress markers: pPERK, pEIF2α, pIRE1α, their respective unphosphorylated proteins, sXBP1, CHOP, and β-actin as a loading control. Representative immunoblots are shown. b Bar graphs represent pPERK/PERK, pEIF2α/ EIF2α, pIRE1/IRE1, sXBP1/β-actin, and CHOP/β-actin as means + SEM. *p < 0.05, **p < 0.01 indicate a significant difference between cells treated with Zyflamend and non-treated cells. c-d Markers of autophagy were examined in the same lysates using antibodies against Beclin 1, LC3, I&II, ATG5, ATG7, and β-actin as a loading control. d Bar graphs represent Beclin 1/β-actin, LC3/β-actin, ATG5/β-actin, and ATG7/β-actin as means + SEM. *p < 0.05, **p < 0.01 indicate a significant difference between cells treated with Zyflamend and non-treated cells. e-f Immunoblots of key proteins in autophagy, AMPK, ER stress, and apoptosis signaling in β-TC6 cells treated with 200 μg/ml Zyflamend with and without the pancaspase inhibitor Z-VAD.fmk. Representative immunoblots are shown. f Bar graphs represent pAMPK/AMPK, pPERK/PERK, pEIF2α/EIF2α, pIRE1α/ IRE1α, sXBP1/β-actin, CHOP/β-actin, pJNK/JNK, Beclin 1/β-actin, LC3I&II/β-actin, and cleaved caspase3/β-actin as means + SEM. *p < 0.05, **p < 0.01 indicate a significant difference between cells treated with Zyflamend and non-treated cells. †p < 0.05, † †p < 0.01 indicate a significant difference between cells treated with Z-VAD.fmk and non-treated cells an adjuvant capable of enhancing the effectiveness of standard cancer therapies.

Discussion
Pancreatic cancer is one of the most untreatable forms of malignancy. The abysmal survival rates for pancreatic cancer diagnosis, leave patients seeking alternatives. The success of current therapies has been limited, and in many cases, ineffective. In addition, these therapies often rely on invasive procedures and high doses that can lead to cytotoxicity and collateral side effects. The need for innovative solutions to these problems has never been higher, and the search for more effective therapies persists. Plant-based compounds are widely considered a promising avenue for combined and synergistic enhancement of known therapies [45,46].
Emerging evidence supports the anti-tumor and proapoptotic effects of Zyflamend on various types of cancers such as prostate [13], melanoma [14], and oral cancer [15]. In this study, we uncover novel insight into the molecular mechanisms through which Zyflamend could prove beneficial both alone and as an adjunct therapy in targeting pancreatic cancer. We demonstrate that  Zyflamend is capable of inducing cell death and reducing cell survival in a time-and dose-dependent manner. Furthermore, we demonstrate that Zyflamend induces ER stress, which in turn activates the JNK pathway leading to autophagy and caspase-mediated cell death (Schema 1). Additionally, Zyflamend enhances the effects of streptozotocin and adriamycin on cell death and survival, two chemotherapeutic compounds used to treat PNETs. Taken together, these findings promote Zyflamend as a potential adjuvant to PNETs therapy. Our findings are in line with Zyflamend cancer research across a broad spectrum of experimental platforms [13-15, 21, 22, 24]. Research has revealed that the individual extracts and constituents contained within Zyflamend could possess therapeutic potential and value as backed by a large number of investigations. For instance, curcumin, a polyphenol found in turmeric, has shown promise in a wide variety of cancer research [17,49]. Curcumin could inhibit carcinogenesis through a variety of proposed pathways and processes including, the inhibition of angiogenesis and tumorigenesis [50], the disruption of the mTOR-raptor complex and subsequently decreased phosphorylation and activation of AKT/mTOR signaling cascade [51,52]. In addition, curcumin can lead to the inactivation of the PI3K/AKT pathway and subsequently induce apoptosis through increased mitochondrial P53 and BCL2-associated X protein (BAX) translocation [53]. Furthermore, curcumin can also regulate autophagy turnover in an AMPK/ ULK1 dependent manner [49]. Moreover, curcumin can induce cell cycle arrest and apoptosis supporting its potential as an adjuvant therapy through increasing the sensitivity of multidrug resistant pancreatic cancer tumors to gemcitabine treatment [54].
Likewise, ginger (another component of Zyflamend) and its derivatives have been evaluated for their anticancer properties [55,56] and were shown to modulate major signaling pathways important for cancer survival and proliferation in several cancer models. Indeed, ginger and its derivatives were demonstrated to induce cancer cell death through modulation of the NF-κB, PI3K/AKT/mTOR, inflammation, cell cycle, angiogenesis, and apoptotic signaling pathways [57,58]. In pancreatic cancer research, ginger and its major pungent components (gingerol, shogaol, and zerumbone) were reportedly shown to downregulate and/or inhibit NF-κB signaling across a variety of pancreatic cancer cell lines [58,59]. For example, 6-shogaol was demonstrated to sensitize PANC-1 cells to gemcitabine through inhibiting NF-κB, cyclin D1, Bcl-2, COX-2, resulting in increased apoptosis [60]. While a majority of the studies have examined the effects of phytochemicals on PDAC, investigations targeting their effects on PNETs remains limited. For instance, isolated polyphenols of rosemary, such as carnosic acid and rosmarinic acid, were shown to cause an increase in TNFα and nitrate levels leading to the induction of apoptosis in rat insulinoma (RINm5F) cells [61,62]. Supportively, treatment of pancreatic endocrine HP62 cells with the phytochemical Devil's club Oplopanax horridus (DC), a close relative of the ginseng family, induced ER stress and apoptosis and exacerbated the anti-proliferative effects of gemcitabine, cisplatin, and paclitaxel [63]. Likewise, carnosic acid RE derivatives also exhibited tumor suppressive potential in a PANC-1 model of PDAC [64]. Careful consideration of the overall biological implications of natural compounds targeting PDAC and PNETs may reveal critical insight into pancreatic cancer oncogenesis, allowing for therapeutic enhancement.
†p < 0.05, † †p < 0.01 indicate a significant difference between cells treated with SP600125 and non-treated cells. c Bar graphs represent the intensity of SRB staining reflective of the cell number and presented as means + SEM from at least three independent experiments. *p < 0.05, **p < 0.01 indicate a significant difference between cells treated with Zyflamend and non-treated cells. †p < 0.05, † †p < 0.01 indicate a significant difference between cells treated with SP600125 and non-treated cells. d-e Colony formation assay. e Bar graphs represent the relative number of colonies in each condition determined by dividing the number of colonies for a given treatment by the total number of colonies in DMSO treated cells (Ctrl.) and expressed as a percentage. f-g Chromatin condensation in cells treated with 200 μg/ml of Zyflamend alone or in combination with SP600125 for 24 h . Representative images are shown. Scale bar: 50 μm. g Bar graphs represent the number of apoptotic cells (Hoechst positive) as means + SEM from at least three independent experiments. In E and G, **p < 0.01 indicates a significant difference between cells treated with Zyflamend and non-treated cells. † †p < 0.01 indicates a significant difference between non-treated and cells treated with SP600125 cancer research models [61,67] through several mechanisms. Petiwala and colleagues demonstrated that rosemary extracts activate the ER stress response and induced apoptosis in 22Rv1 and LNCaP prostate cancer cells. In these cell lines, the rosemary extracts also increased the expression of BAX, cleaved caspase-3, CHOP, and IRE1α, in a mechanism similar to our findings [68] Finally, the Zyflamend component holy basil has also shown promise in pancreatic cancer research as it inhibited tumorigenesis in both murine and in vitro models and promoted apoptosis [69]. Taken together, these findings demonstrate the potential for combining these herbal extracts to target pancreatic cancer. The development and progression of pancreatic cancer have been linked to the activation and inhibition of a variety of cell signaling pathways. In this study, we explored the role of Zyflamend on cell survival, cell cycle, and cell death. We demonstrate that Zyflamend attenuates cell survival, causes G2/M cell cycle arrest and promote apoptotic cell death in a dose and time dependent manner (Schema 1). While 800 μg/ml completely inhibited cell growth, 200 μg/ml was sufficient to reduce cell survival significantly. Based on previously described findings, we chose 200 μg/ml for further exploration because this dose represents the maximum plasma concentration of one of Zyflamend's constituents, curcumin, in humans following supplementation [18]. Importantly, previous studies have revealed that bioactive compounds such as those found in Zyflamend, can be more effective when combined. Notably, compounds exhibiting relatively low bioactivity in isolation often show increased activation when combined with another bioactive originating from the same source [18]. Moreover, Zyflamend doses at or below 200 μg/ml have proven effective, to varying degrees, in either inducing apoptosis and/or inhibiting proliferation of several tumor-derived cell lines, including pancreatic [22] and prostate [70] cancer cells. In agreement with these studies, we report here that this dose was sufficient to significantly induce apoptotic cell death in pancreatic cancer cells. On the other hand, while the individual components of Zyflamend show potential in targeting cancer, the doses often required to elicit responses exceed physiological relevance.
For instance, curcumin dose required to elicit the inhibition of AKT phosphorylation occurred at high doses (> 40 μM) in a previously mentioned study [51]. In addition, the dose required to generate an anti-tumor response to a polyphenolic extract from ginger, 6-shogaol, was relatively high, both in vitro (15-20 uM) and in vivo (50 mg/ Kg) in a pancreatic cancer model [60]. Research on rosemary extracts shows significant variation in the doses used in cancer research with in vivo doses reported from a low of 100 mg/Kg/day to as high as 3333.3 mg/Kg/day [61]. Furthermore, a dose of 6 g of green tea per day given to prostate cancer patients led to varying degrees of collateral cytotoxicity [71]. In order to develop translationally valid phytochemical research, it is essential to carefully consider several factors including the human equivalent dose, the clinical validity of the experimental model, and the molecular mechanisms involved.
In order to determine the molecular mechanisms mediating Zyflamend's actions, we explored pathways that are critical for pancreatic cancer cell survival and proliferation. We found that Zyflamend induced the cleavage of caspase-3 along with its downstream target PARP. This finding is in alignment with other studies showing similar activation of the apoptotic cascade in response to Zyflamend treatment in colon, prostate, pancreatic (Panc-28) and bladder cancer models [23,25]. Next, we probed for changes in the MAPK pathways in response to Zyflamend. As aforementioned, the MAPK pathways include MEK1/ERK1/2/p90 RSK, P38 MAPK, and JNK/ c-JUN pathways. In conjunction with the PI3K/AKT/ mTOR pathway, these proteins and pathways are responsible for key physiological responses such as cell survival, protein synthesis, cell proliferation, growth, migration, and apoptosis in both normal and cancerous cells [72,73]. In addition, elevated activation of the PI3K signaling cascade is observed in a majority of cancers. Therefore, targeting the PI3K pathway with natural compounds could provide invaluable therapeutic potential to cancer patients. At much higher doses, individual components found in Zyflamend such as curcumin, ginger (6-shogaol) [74], and rosemary [16] have all been shown (See figure on previous page.) Fig. 6 Zyflamend Exacerbates the Effects of Streptozotocin and Adriamycin on ER Stress, Autophagy, and Cell Death in β-TC6 Cells. Total cell lysates from control cells treated with Zyflamend and non-treated cells with or without streptozotocin (STZ; 5 mM) or adriamycin (ADR; 5 μM) for 24 h were immunoblotted for markers of ER stress (a-b), autophagy, and cell death (c-d). Representative immunoblots are shown. b Bar graphs represent pPERK/PERK, pEIF2α/EIF2α, pIRE1/IRE1, sXBP1/β-actin, and CHOP/β-actin as means + SEM. d Bar graphs represent Beclin 1/β-actin, LC3/ β-actin, ATG5/β-actin, ATG7/β-actin, and cleaved caspase-3 / β-actin as means + SEM. e-f Colony formation assay. f Bar graphs represent the relative number of colonies in each condition determined by dividing the number of colonies for a given treatment by the total number of colonies in DMSO treated cells (Ctrl.) and expressed as a percentage. g Cell toxicity assay using the MTT method. Bar graphs represent the intensity of formazan staining reflective of the cell number and presented as means + SEM. In b, d, f and g, *p < 0.05, **p < 0.01 indicate a significant difference between cells treated with Zyflamend and non-treated cells. †p < 0.05, † †p < 0.01 indicate a significant difference between cells treated with Zyflamend combined with the chemotherapeutic agents (streptozotocin or adriamycin) and cells treated with streptozotocin or adriamycin only to inhibit the phosphorylation of AKT and cell survival of various cancer types. In addition, curcumin has been shown to inhibit the phosphorylation of AKT in a pancreatic cancer cell line (PANC-1) [75]. In line with these findings, our data demonstrate that Zyflamend treatment caused a significant reduction in both ERK1/2 and AKT phosphorylation.
A key finding from this research is that, in addition to inducing autophagy, Zyflamend also upregulates ER stress and through this could reduce the survival of pancreatic cancer cells. The ER stress response is a set of highly regulated mechanisms sensitive to a wide variety of physiological and pathological conditions that affect protein synthesis and folding. One of the mechanisms through which misfolded and/or unfolded proteins are handled by the ER is through ER-associated protein degradation (ERAD). As improperly folded aggregated proteins reach excessive thresholds, the primary functions of the ER stress response can be overwhelmed. To cope with this rising stress level, the autophagy-lysosome dependent ERAD (type II) machinery can be activated [76,77]. If the level of unfolded proteins continues to rise, the stress can overwhelm both autophagy and the ER's capacity to function, leading to the induction of programmed cell death [77]. In the environment of tumorigenesis, the elevated levels of metabolic stress can often lead to the activation of autophagy and ER stress responses [78,79]. The findings of Klieser et al., in BON1 cells, support the potential in targeting ER stress as a therapeutic approach to PNETs [80]. However, the activation of autophagy and the proper functioning of ER stress responses act in duality where properly functioning can protect cancer from the harsh microenvironment, and when critical thresholds are reached, it can initiate cell death [79]. In this study, we demonstrated that Zyflamend treatment led to the activation of two branches of the ER stress mediated unfolded protein response (UPR) through increased phosphorylation of PERK and IRE1α and activation of their downstream targets EIF2a and sXBP1. In addition, this was accompanied by elevated levels of CHOP, an ER stress mediated apoptosis inducer. Taken together, our findings show that Zyflamend can act as an inducer of apoptotic cell death through modulation of ER stress and autophagy and support the hypothesis of the potential beneficial effect of targeting ER stress and autophagy for the treatment of PNETs. In recent times, both natural and synthetic compounds have been explored regarding ER stress and autophagy mediated apoptosis in various cancer models [81]. However, this study is the first to delineate how natural compounds can act synergistically in the coordination of ER stress, MAP kinases, and autophagy signaling to induce cell death in pancreatic cancer cells.

Conclusions
In summary, our study shows that, through a unique combination of 10 herbs, the therapeutic potential for targeting PNETs (and possibly other types of cancer) could be enhanced in an additive or synergistic manner. It is our hope, the findings from this study may pave the way towards pre-clinical validation of the beneficial effects of Zyflamend as a potential adjuvant for the treatment for PNETs. It is also our hope that finding from this study could be soon translated into clinical trials aimed at developing safer and more potent therapeutic approaches capable of improving pancreatic cancer patient outcomes and quality of life.
Additional file 2: Supplementary Fig. 1. Zyflamend Causes Toxicity and Exacerbates the Effects of Streptozotocin and Adriamycin on ER Stress, Autophagy, and Cell Death in RIN-5F Cells. A-B) Colony formation assay. B) Bar graphs represent the relative number of colonies in each condition determined by dividing the number of colonies for a given treatment by the total number of colonies in DMSO treated cells (Ctrl.) and expressed as a percentage. C) Cell toxicity assay using the MTT method. Bar graphs represent the intensity of formazan staining reflective of the cell number and presented as means + SEM. D-G) Total cell lysates from control cells treated with Zyflamend and non-treated cells with or without streptozotocin (STZ; 5 mM) or adriamycin (ADR; 5 μM) for 24 h were immunoblotted for markers of ER stress (D-E), autophagy, and cell death (F-G). Representative immunoblots are shown. E) Bar graphs (See figure on previous page.) Schema 1 Overview of the Pro-Apoptotic Effects of Zyflamend in Pancreatic Cancer Cells. Zyflamend treatment induced ER stress, autophagy, MAP kinases and apoptosis in pancreatic β-TC6 Cells. Previous studies have shown that activation of AMPK in pancreatic cancer cells by chemotherapeutic agens induces apoptosis and autophagy via the AMPK/mTOR signaling pathway (dotted lines) [47]. Similarly, activation of the JNK/SAPK pathway [48] by stressors and chemotherapeutic agents can lead to apoptotic cell death of pancreatic cancer cells. Our study shows that treatment with pan-caspase inhibitor Z-VAD.fmk prevented the cleavage of caspase 3 but had no effects on AMPK, autophagy, or ER stress. Zyflamend treatment activated AMPK; however, treatment with AMPK inhibitor CC did not result in changes to ER stress, apoptosis, autophagy, or JNK signaling pathways. Autophagy inhibition with 3-MA inhibited autophagy and apoptosis only. Zyflamend activated both the IRE-1 and PERK branches of the ER stress mediated UPR and resulted in the subsequent upregulation of the downstream JNK signaling pathway. ER stress inhibition with 4-PBA resulted in decreased JNK activation, autophagy, and apoptosis. While inhibition of JNK with SP600125 significantly reduced the the level of both, apoptosis and autophagy represent pPERK/PERK, pEIF2α/EIF2α, pIRE1/IRE1, sXBP1/β-actin, and CHOP/β-actin as means + SEM. *p < 0.05, **p < 0.01 indicate a significant difference between cells treated with Zyflamend and non-treated cells. G) Bar graphs represent Beclin 1/β-actin, LC3/β-actin, ATG5/β-actin, ATG7/β-actin, and cleaved caspase-3 / β-actin as means + SEM. In C, E, and G *p < 0.05, **p < 0.01 indicate a significant difference between cells treated with Zyflamend and non-treated cells. †p < 0.05, † †p < 0.01 indicate a significant difference between cells treated with Zyflamend combined with the chemotherapeutic agents (streptozotocin or adriamycin) and cells treated with streptozotocin or adriamycin only.