Achiral Mannich-Base Curcumin Analogs Induce Unfolded Protein Response and Mitochondrial Membrane Depolarization in PANC-1 Cells

Achiral Mannich-type curcumin analogs have been synthetized and assayed for their cytotoxic activity. The anti-proliferative and cytotoxic activity of curcuminoids has been tested on human non-small-cell lung carcinoma (A549), hepatocellular carcinoma (HepG2) and pancreatic cancer cell line (PANC-1). Based on the highest anti-proliferative activity nine drug candidates were further tested and proved to cause phosphatidylserine exposure as an early sign of apoptosis. Curcumin analogs with the highest apoptotic activity were selected for mechanistic studies in the most sensitive PANC-1 cells. Cytotoxic activity was accompanied by cytostatic effect since curcumin and analogs treatment led to G0/G1 cell cycle arrest. Moreover, cytotoxic effect could be also detected via the accumulation of curcuminoids in the endoplasmic reticulum (ER) and the up-regulation of ER stress-related unfolded protein response (UPR) genes: HSPA5, ATF4, XBP1, and DDIT3. The activated UPR induced mitochondrial membrane depolarization, caspase-3 activation and subsequent DNA breakdown in PANC-1 cells. Achiral curcumin analogs, C509, C521 and C524 possessed superior, 40-times more potent cytotoxic activity compared to natural dihydroxy-dimetoxycurcumin in PANC-1 cells.


Introduction
Turmeric spice obtained from the rhizomes of Curcuma longa has been used for centuries in traditional Chinese medicine and in Ayurveda in India based on its assumed anti-inflammatory, anti-microbial, and anti-cancer effects [1]. Curcuminoids make up 2-5% of turmeric powder and consist of dihidroxy-dimetoxycurcumin (curcumin, 77%), mono-demethoxycurcumin (17%) and bis-demethoxycurcumin (3%) [2]. Due to the empirical beneficial health effect of turmeric the consumption of curcumin as a dietary supplement increased worldwide. By today, curcumin has been designated "Generally Recognized as Safe (GRAS)" by the Food and Drug Administration of the United States [3]. The widespread consumption of turmeric and curcumin and its potential as a pharmaceutical laid down the basis for the need of proper scientific studies to reveal the effects of curcumin on human health as well as the design and synthesis of curcumin analogs which can be potentially introduced to the clinic. Our aim was to increase the cytotoxicity of Mannich-type curcumin analogs and reveal their mechanism of action in a cellular system.
Tumorigenesis is a multistep process in which curcumin has been implicated to suppress the growth of tumor cells by reducing their proliferation and survival as well as to induce tumor cell death [2,4,5]. It has been published that curcumin inhibited the proliferation and induced the apoptosis of non-small-cell lung carcinoma cells (A549 cell line) by the up-regulation of microRNA-192-5p and suppression of the PI3K3/Akt signaling pathway [6]. In another study apoptosis of A549 cells induced by curcumin relied on oxidative stress and Mitogen-activated protein kinase (MAPK) pathway [7]. Curcumin attenuate not only tumor cell viability but also the metastatic ability of A549 cells by blocking the adiponectin receptor 1 [8] and glucose transporter 1 (GLUT1) [9]. Therefore, we tested our novel curcumin analogs on the non-small cell lung cancer (NSCLC) cell line, A549.
Curcumin caused apoptosis of human hepatocellular carcinoma HepG2 cells as well, via disruption of mitochondrial membrane potential [10] and counteracted fatty acid synthase [11]. In our study, we tested our analogs on both A549 cells as well as on the hepatocellular carcinoma (HCC) cell line HepG2.
Finally, we focused on PANC-1 cells, because among solid tumors, pancreatic malignancy has the highest mortality rate with the overall 5-year survival being less than 6% [12]. The possible application of curcumin in pancreatic cancer has been reported earlier where the authors showed that PANC-1 cells were sensitive to curcumin treatment via reduction of the inhibitors of apoptosis [13] and through the induction of forkhead box O1 [14].
Here, we present achiral Mannich-type curcumin analogs with potential cytotoxic activity on human NSCLC, hepatocellular carcinoma, and pancreatic cancer cell lines.

Achiral Curcumin Analogs Impaired Tumor Cell Viability
Recently, we have reported the potent cytotoxic activity of racemic Mannich-type curcumin analogs [15][16][17]. Here, we report the cytotoxic activity of the achiral curcumin analog library (C500 series) (Figure 1 and Figure S1).  The substitution pattern was carefully chosen based on the already established structure-activity relationship (SAR) results [17], with the ultimate aim to design a simpler and achiral library of curcuminoids with an improved pharmacokinetic profile. All intermediates and compounds were synthesized based on the previously established method [17,18]. As a pre-screening we tested the anti-proliferative effect of the first analog, C501 by measuring the confluency by live cell analyzer of C501 treated A549 cells ( Figure S2). Since the anti-proliferative activity of C501 reached its maximum effect at 3.125 µM, we used lower concentrations of the members of C500 series (0. 16-5 µM) in the subsequent experiments. We defined the dose-response curves of the 23 members of C500 series by the significantly more sensitive resazurin assay on A549, HepG2, and PANC-1 cells ( Figure S3). The half maximal inhibitory concentration (IC 50 ) values are presented in Table 1. The IC 50 value of curcumin was above the pharmacologically favorable range 27.11 µM, 14.53 µM, and 30.57 µM in A549, HepG2, and PANC-1 cells, respectively. Six molecules were completely inactive at the applied concentrations (C514, C520, C525, C526, C529, and C530) and were not tested further. This was in full accordance with previously established SAR results [17,18], that the presence of functionalities such as carboxylic acid, dihydroxyphenyl, or para-hydroxy substituents resulted in the complete loss of cytotoxic activity. Compounds which possessed low anti-proliferative activity (IC 50 > 5 µM) were also excluded from further testing (C502, C510, C513, C516, C517, C519, C532, and C533). The current project focused on curcumin analogs with potent cytotoxic activity on three different cell lines: A549, HepG2, and PANC-1. Nine of the 23 novel curcumin analogs (C501, C503, C504, C505, C509, C515, C518, C521, and C524) hampered the viability of all three tested cell lines when used below 5 µM. In order to clarify whether the viability of A549, HepG2, and PANC-1 cells was hampered by apoptosis or necrosis, we carried out annexin V-propidium iodide (PI) staining on the cells treated by the nine selected candidates. Curcumin and its analogs showed dose dependent phosphatidylserine exposure that suggests potent apoptotic activity (AnnV + /PI early apoptosis and AnnV + /PI + late apoptosis) in A549 (Figure 2A), HepG2 ( Figure 2B), and PANC-1 cells ( Figure 2C) without the appearance of a massive, only PI positive necrotic population ( Figure S4). described in Section 3.2. Materials and Methods. Hashmark ( # ) labeled curcumin analogs were selected for further investigation.

Curcumin Analogs Induced Phosphatidylserine Exposure of A549, HepG2 and PANC-1 Cells
In order to clarify whether the viability of A549, HepG2, and PANC-1 cells was hampered by apoptosis or necrosis, we carried out annexin V-propidium iodide (PI) staining on the cells treated by the nine selected candidates. Curcumin and its analogs showed dose dependent phosphatidylserine exposure that suggests potent apoptotic activity (AnnV + /PI⁻ early apoptosis and AnnV + /PI + late apoptosis) in A549 (Figure 2A), HepG2 ( Figure 2B), and PANC-1 cells ( Figure 2C) without the appearance of a massive, only PI positive necrotic population ( Figure S4). In order to identify the most active analogs, we summarized the percentage of total apoptotic cell populations at the lowest applied concentration for each analog in Table 2. Three compounds stood out with appreciable activity, C509, C521, and C524, therefore only these were included in In order to identify the most active analogs, we summarized the percentage of total apoptotic cell populations at the lowest applied concentration for each analog in Table 2. Three compounds stood out with appreciable activity, C509, C521, and C524, therefore only these were included in further experiments. All three molecules share the common C-4 chloroacetamidomethyl and either the meta-hydroxy or methoxy substituents on their side-chains. An analog with moderate activity, was also included (C501) in our analyses. Since PANC-1 cells were the most sensitive in our study, additionally the 5-year overall survival has been reported to be the lowest for pancreatic cancer (less than 6%) among other solid malignancies [12], we used PANC-1 cells for subsequent mechanistic studies to reveal how novel curcumin analogs counteract cancer cell viability.
According to several reports it is unequivocal that curcumin can dysregulate the cell cycle. It has been reported that natural curcumin caused G 2 /M mitotic catastrophy, in relation to the G 2 /M phase in head and neck squamous cell carcinoma or in bovine aortic endothelial cells after 24 h incubation [19,20], it was also shown that curcumin caused not only G 1 /S but also G 0 /G 1 cell cycle arrest in human prostate cancer cells [21,22]. The differences in cell cycle dysregulation may be cell type specific or may depend on the different experimental conditions, incubation time period, the concentration and formulation of curcumin. the meta-hydroxy or methoxy substituents on their side-chains. An analog with moderate activity, was also included (C501) in our analyses. Since PANC-1 cells were the most sensitive in our study, additionally the 5-year overall survival has been reported to be the lowest for pancreatic cancer (less than 6%) among other solid malignancies [12], we used PANC-1 cells for subsequent mechanistic studies to reveal how novel curcumin analogs counteract cancer cell viability.
According to several reports it is unequivocal that curcumin can dysregulate the cell cycle. It has been reported that natural curcumin caused G2/M mitotic catastrophy, in relation to the G2/M phase in head and neck squamous cell carcinoma or in bovine aortic endothelial cells after 24 h incubation [19,20], it was also shown that curcumin caused not only G1/S but also G0/G1 cell cycle arrest in human prostate cancer cells [21,22]. The differences in cell cycle dysregulation may be cell type specific or may depend on the different experimental conditions, incubation time period, the concentration and formulation of curcumin.

Curcumin Analogs Induced ER (Endoplasmic Reticulum) Stress and Mitochondrial Membrane Depolarization
Among others, we have previously shown that perturbing the homeostasis of the ER could reduce cellular viability [23]. It was recently published that natural curcumin caused ER stress mediated apoptosis in cervical cancer cells [24] and in A549 cells [7]. In order to clarify whether the apoptotic effect of curcumin analogs in PANC-1 cells relied on the ER stress related mitochondrial apoptotic pathway, we followed the accumulation of the tested agents. Since curcumin possesses inherent fluorescence [25] and our analogs retained this property, we tested the subcellular localization of curcumin and our analogs in PANC-1 cells by fluorescence confocal microscopy. Similarly to curcumin, our analogs localized in the ER which was further verified by ER tracker co-localization ( Figure 4).

Curcumin Analogs Induced ER (Endoplasmic Reticulum) Stress and Mitochondrial Membrane Depolarization
Among others, we have previously shown that perturbing the homeostasis of the ER could reduce cellular viability [23]. It was recently published that natural curcumin caused ER stress mediated apoptosis in cervical cancer cells [24] and in A549 cells [7]. In order to clarify whether the apoptotic effect of curcumin analogs in PANC-1 cells relied on the ER stress related mitochondrial apoptotic pathway, we followed the accumulation of the tested agents. Since curcumin possesses inherent fluorescence [25] and our analogs retained this property, we tested the subcellular localization of curcumin and our analogs in PANC-1 cells by fluorescence confocal microscopy. Similarly to curcumin, our analogs localized in the ER which was further verified by ER tracker colocalization ( Figure 4).  The subcellular localization of curcumin and its analogs was assessed with endoplasmic reticulum (ER) tracker co-localization by laser scanning confocal microscopy. To further minimize channel crosstalk, only curcumin analog labeled samples were also prepared (inset images) and used as reference for image capturing conditions for curcumin-ER tracker dual labelled samples. Representative images are shown. Scale bar at C524 inset image is valid for all insets (controls). Scale bar at the lower right corner of C524 is valid for all ER co-localization images. Both scale bars are 20 µm.
The subcellular localization of curcumin analogs in the ER may activate an adaptive response to ER stress known as the unfolded protein response (UPR), which up-regulates ER chaperons, halts translation of secretory proteins, and degrades misfolded proteins. If the ER stress is irreversible the UPR mediates apoptotic cell death to restore the cell homeostasis [26]. ER stress was monitored by the induction of UPR related genes: HSPA5 (Heat Shock Protein Family A (Hsp70) Member 5), ATF4 (Activating Transcription Factor 4), XBP1 (X-Box Binding Protein 1), and DDIT3 (DNA Damage Inducible Transcript 3). HSPA5 is a chaperon, master regulator of the UPR [27], ATF4, and XBP1 are transcriptional activators of UPR genes and chaperons [28], DDIT3 (encoding CHOP) is a transcription factor mediating ER stress related apoptosis [17,26,29].
Curcumin and the tested analogs induced the expression of all tested genes 12 h after treatment ( Figure 5). The up-regulation of HSPA5 was two-fold by 12.5 µM curcumin and around 10-fold by 1.25 µM C509, C521, and C524 treatment. ATF4 and XBP1 were around 4-5-fold up-regulated upon curcumin and curcumin analog stimulation. The highest increase was detected in case of DDIT3, where a 13-fold increase was detected with curcumin (25 µM), while the analogs induced around 30-fold overexpression at 1.25 µM, only C501 showed 60-fold change at 5 µM. DDIT3 overexpression may suggest that in curcumin and curcumin analog treated cells the UPR progressed to a state where homeostasis cannot be restored and the cells are committed to an apoptotic fate [29,30]. The concentration dependent decline of gene expression changes (50 µM curcumin and 5 µM C509, C521, and C524) may be due to the advanced apoptotic program. The subcellular localization of curcumin analogs in the ER may activate an adaptive response to ER stress known as the unfolded protein response (UPR), which up-regulates ER chaperons, halts translation of secretory proteins, and degrades misfolded proteins. If the ER stress is irreversible the UPR mediates apoptotic cell death to restore the cell homeostasis [26]. ER stress was monitored by the induction of UPR related genes: HSPA5 (Heat Shock Protein Family A (Hsp70) Member 5), ATF4 (Activating Transcription Factor 4), XBP1 (X-Box Binding Protein 1), and DDIT3 (DNA Damage Inducible Transcript 3). HSPA5 is a chaperon, master regulator of the UPR [27], ATF4, and XBP1 are transcriptional activators of UPR genes and chaperons [28], DDIT3 (encoding CHOP) is a transcription factor mediating ER stress related apoptosis [17,26,29].
Curcumin and the tested analogs induced the expression of all tested genes 12 h after treatment ( Figure 5). The up-regulation of HSPA5 was two-fold by 12.5 µM curcumin and around 10-fold by 1.25 µM C509, C521, and C524 treatment. ATF4 and XBP1 were around 4-5-fold up-regulated upon curcumin and curcumin analog stimulation. The highest increase was detected in case of DDIT3, where a 13-fold increase was detected with curcumin (25 µM), while the analogs induced around 30fold overexpression at 1.25 µM, only C501 showed 60-fold change at 5 µM. DDIT3 overexpression may suggest that in curcumin and curcumin analog treated cells the UPR progressed to a state where homeostasis cannot be restored and the cells are committed to an apoptotic fate. [29,30]. The concentration dependent decline of gene expression changes (50 µM curcumin and 5 µM C509, C521, and C524) may be due to the advanced apoptotic program.  The perturbation of the homeostasis of the ER by curcumin analogs may confound the subcellular oxidative homeostasis affecting mitochondrial membrane potential (MMP) [31]. Therefore, mitochondrial membrane depolarization was examined by JC-1 (5,5 ,6,6 -tetrachloro-1,1 ,3,3tetraethylbenzimidazolocarbocyanine iodide) staining following curcumin and curcumin analog treatment of PANC-1 cells. Not only curcumin but also our analogs depolarized mitochondria of PANC-1 cells in a concentration dependent manner ( Figure 6). The effect of curcumin on MMP was significant at 50 µM, while C501 analog was effective at 5 µM, C509 at 2.5 µM, and C521 and C524 at 1.25 µM. in PANC-1 cells treated with curcumin and curcumin analogs after 12 h. Gene expression was analyzed by qRT-PCR (quantitative real-time polymerase chain reaction) as described in the Section 3.5. Materials and methods, * p < 0.05, ** p < 0.01, *** p < 0.001.

Curcumin Analogs Induced Caspase-3 Activation and DNA Fragmentation
Mitochondrial membrane depolarization may lead to the activation of caspases which renders cells for apoptosis [32]. Effector caspase-3 activation occurred in PANC-1 cells following curcumin and curcumin analog treatment in a concentration dependent manner (Figure 7). Among curcumin analogs C524 showed the highest induction of caspase-3 at 5 µM.

Curcumin Analogs Induced Caspase-3 Activation and DNA Fragmentation
Mitochondrial membrane depolarization may lead to the activation of caspases which renders cells for apoptosis [32]. Effector caspase-3 activation occurred in PANC-1 cells following curcumin and curcumin analog treatment in a concentration dependent manner (Figure 7). Among curcumin analogs C524 showed the highest induction of caspase-3 at 5 µM.
Apoptosis executed by the activation of caspases results in the internucleosomal degradation of genomic DNA which can be studied by flow cytometry as a hypodiploid sub-G 1 fraction of the cell population [33]. In order to confirm that event we analyzed the percentage of cells with degraded DNA content ( Figure 8A). Using PANC-1 cells, our results showed that treatment with curcumin resulted in DNA degradation at 25 µM, while curcumin analogs exerted a significant effect at 1.25 µM. Cytotoxicity of the curcumin analogs resulted in DNA breakdown as the final step of apoptosis in a concentration dependent manner ( Figure 8B). Apoptosis executed by the activation of caspases results in the internucleosomal degradation of genomic DNA which can be studied by flow cytometry as a hypodiploid sub-G1 fraction of the cell population [33]. In order to confirm that event we analyzed the percentage of cells with degraded DNA content ( Figure 8A). Using PANC-1 cells, our results showed that treatment with curcumin resulted in DNA degradation at 25 µM, while curcumin analogs exerted a significant effect at 1.25 µM. Cytotoxicity of the curcumin analogs resulted in DNA breakdown as the final step of apoptosis in a concentration dependent manner ( Figure 8B).   Apoptosis executed by the activation of caspases results in the internucleosomal degradation of genomic DNA which can be studied by flow cytometry as a hypodiploid sub-G1 fraction of the cell population [33]. In order to confirm that event we analyzed the percentage of cells with degraded DNA content ( Figure 8A). Using PANC-1 cells, our results showed that treatment with curcumin resulted in DNA degradation at 25 µM, while curcumin analogs exerted a significant effect at 1.25 µM. Cytotoxicity of the curcumin analogs resulted in DNA breakdown as the final step of apoptosis in a concentration dependent manner ( Figure 8B).

Cell Culturing and Treatments
Cells were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia, USA). A549, HepG2 cells were maintained in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) 10% fetal calf serum (FCS, Gibco) and PANC-1 cells were maintained in Roswell Park Memorial Institute 1640 medium (RPMI-1640) 10% FCS, pH of the cell culture media was controlled to be between 7.2-7.4 prior use. Media were supplemented with 2 mM GlutaMAX, and 100 U/mL penicillin, 100 µg/mL streptomycin (Life Technologies, Carlsbad, California, USA). The cell cultures were maintained at 37 • C in a humidified incubator in an atmosphere of 5% CO 2 (Sanyo, Japan).
Curcumin and curcumin analogs were dissolved in dimethyl sulfoxide (DMSO) at 10 mM concentration. Since DMSO can be toxic for cellular systems, the stock solution was further diluted in serial dilutions in all cases in the appropriate cell culture media. The intermediate dilution of curcumin was 250 µM (40× dilution), it was serially diluted to 125 µM (80× dilution) and it was further diluted to 62.5 µM (160× dilution), then each intermediate dilution was further diluted 5× when it was added to the cells, so the treatments of curcumin were 50 µM (200×), 25 µM (400×) and 12.5 µM (800×). The intermediate dilution of curcumin analogs was 25 µM (400×), it was serially diluted to 12.5 µM (800× dilution) and it was further diluted to 6.25 µM (1600× dilution), then each intermediate dilution was further diluted 5× when it was added to the cells so the treatments of curcumin analogs were 5 µM (2000×), 2.5 µM (4000×), and 1.25 µM (8000×).

Resazurin Viability Assay
The viability of A549, HepG2, and PANC-1 cells was determined by the fluorescent resazurin assay as described previously [34]. Briefly, cells (6 × 10 3 ) were seeded into 96-well plates (Corning Life Sciences, Tewksbury, MA, USA) in media. Cells were cultured overnight before treatment. Viability was determined after a 72 h incubation in treated and non-treated cells. Resazurin reagent (Sigma-Aldrich) was dissolved in phosphate buffered saline (PBS, pH 7.4) at 0.15 mg/mL concentration, sterile filtered (0.22 µm, Merck Millipore) and aliquoted at −20 • C. Samples were treated in 120 µL volume with a final concentration of 25 µg/mL resazurin. After 2 hours incubation at 37 • C 5% CO 2 , fluorescence (530 nm excitation/580 nm emission) was recorded on a multimode microplate reader (Cytofluor4000, PerSeptive Biosytems, Framingham, MA, USA). Viability was calculated in relation to untreated control cells and blank wells containing media without cells. The presented IC 50 values (half maximal inhibitory concentration) were determined based on dose-response curves plotted in GraphPad Prism ® 5 (La Jolla, CA, USA).

Detection of Phosphatidylserine Exposure
Cells (5 × 10 4 ) were plated in 24-well tissue culture plates (Corning Life Sciences) and treated in 500 µL volume with increasing concentrations of test compounds in duplicates. After 72 h the supernatants were harvested. Cells were washed with PBS, trypsinized, pooled with the corresponding supernatant and pelleted by centrifugation (2000 rpm, 5 min). The pellet was resuspended in annexin V binding buffer (0.01 M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 0.14 M NaCl and 2.5 mM CaCl 2 ). Annexin V-Alexa Fluor ® 488 (AnnV, Life Technologies, 2.5:100) was added to the cells, which were then kept in the dark at room temperature for 15 min. Propidium iodide (PI, Sigma-Aldrich, St. Louis, MO, USA) was added at a final concentration of 10 µg/mL right before the acquisition of samples. Cells (2 × 10 4 events) were analyzed on a FACSCalibur flow cytometer using CellQuest software (Becton Dickinson, Franklin Lakes, NJ, USA). The percentage of annexin V-Alexa Fluor ® 488 positive (FL1 channel, 530/30 nm filter) and propidium iodide negative (FL3 channel, 670 nm filter) early apoptotic cells and FL1 positive and FL3 positive late apoptotic cells were determined. The total apoptotic population includes both early and late apoptotic cells. Bar graphs were created by GraphPad Prism ® 5.

Gene Expression Analysis
PANC-1 cells (2 × 10 6 ) were plated in 60 mm tissue culture dishes (Corning Life Sciences) in RPMI 10% FCS. Twenty-four hours later cells were treated in 5 mL volume with test compounds. After 12 h treatment, nucleic acid preparation was done by using the Bioneer RNA purification kit (Bioneer, Viral RNA extraction kit, Daejeon, South Korea) according to an already published protocol [37] with modifications [38]. The quality and quantity of the isolated RNA were measured with NanoDrop1000 Version 3.8.1. (Thermo Fisher Scientific).
Reverse transcription from 3 µg of total RNA was performed with the High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, California, USA) in a total volume of 30 µL according to the manufacturer's protocol. After dilution with 130 µL of ultrapure water (Applied Biosystems) cDNA was used as template for gene expression analysis. Quantitative real-time PCR (qRT-PCR) was performed on the LightCycler®Nano Instrument (Roche, Basel, Switzerland) using gene-specific primers with SYBR Green protocol as described previously [38]. Briefly: for cycling each 10 µL PCR reaction contained 1 µL cDNA, 250 nM primers and 5 µL FastStart Essential DNA Green Master (2×, Roche). Primers were as follows: Heat Shock Protein Family A (Hsp70) Member 5 (HSPA5, NM_005347.4), HSPA5-F 5 -AGCCTGGCGACAAGAGTG-3 , HSPA5-R 5 -TCCTTGGGCAGTATTGGATT-3 ; the following primer sequences are referenced by [17]: Activating Transcription Factor 4 (ATF4, NM_182810. The PCR protocol was as follows: enzyme activation at 95 • C for 10 min, 45 cycles of denaturation at 95 • C for 15 s, annealing at 60 • C and extension at 72 • C for 25 s. All the PCRs were performed with three replicates. After amplification, melting curve was checked to verify the specificity of the PCR reactions. The presented relative gene expression ratios were normalized to ACTB gene, calculated using the comparative CT method (2 −∆∆CT ). Fold change refers to 2 −∆∆Ct treated /2 −∆∆Ct untreated . All values are presented as mean ± standard deviation (SD).

Statistical Analysis
Statistical analysis was performed using two-tailed, homoscedastic Student's t-test to evaluate the statistical significance (set at * p < 0.05, ** p < 0.01, *** p < 0.001) using pairwise comparison of each sample to the untreated control in Microsoft ® Excel ™ .

Conclusions
Despite the emerging field of targeted anti-cancer therapeutics the combination of different therapeutic strategies (surgery, radiation, chemotherapeutic agents, biologics and immunotherapy etc.) may combat cancer and mount an effective therapeutic response [39][40][41][42], however, there is a high need for novel anti-cancer agents with improved cancer cell killing with less side effects.
We designed and synthetized a library of twenty-three achiral curcumin analogs to follow up on our previous work to identify potent, cytotoxic Mannich-base curcuminoids [15,17]. These compounds lack a chiral center so their biological/pharmacological testing is more straightforward without the need for the evaluation of the individual enantiomers. We tested the anti-proliferative activity of our curcumin analogs on human non-small-cell lung carcinoma (A549), hepatocellular carcinoma (HepG2), and pancreatic cancer (PANC-1) cell lines.
Three curcuminoids, C509, C521, and C524 with the highest apoptotic activity were selected for mechanistic studies in the most sensitive PANC-1 cells. We found that the biological activity of the analogs on one hand relied on a cytostatic effect since curcumin and curcuminoid analog treatment led to G 0 /G 1 cell cycle arrest. On the other hand, our compounds also possessed a potent cytotoxic effect (Figure 9). Their accumulation in the ER caused ER stress, the induction of the unfolded protein response, mitochondrial membrane depolarization, caspase-3 activation, and subsequently DNA breakdown.
Three curcumin analogs, C509, C521, and C524 possessed superior, 40-times more potent cytotoxic activity compared to the natural dihydroxy-dimetoxycurcumin in human pancreatic cancer cells, offering the possibility for their further investigation. activity of our curcumin analogs on human non-small-cell lung carcinoma (A549), hepatocellular carcinoma (HepG2), and pancreatic cancer (PANC-1) cell lines. Three curcuminoids, C509, C521, and C524 with the highest apoptotic activity were selected for mechanistic studies in the most sensitive PANC-1 cells. We found that the biological activity of the analogs on one hand relied on a cytostatic effect since curcumin and curcuminoid analog treatment led to G0/G1 cell cycle arrest. On the other hand, our compounds also possessed a potent cytotoxic effect (Figure 9). Their accumulation in the ER caused ER stress, the induction of the unfolded protein response, mitochondrial membrane depolarization, caspase-3 activation, and subsequently DNA breakdown.
Three curcumin analogs, C509, C521, and C524 possessed superior, 40-times more potent cytotoxic activity compared to the natural dihydroxy-dimetoxycurcumin in human pancreatic cancer cells, offering the possibility for their further investigation.