Apoptosis In BxPC-3 Cells Research Articles

Abstract 7308: Induction of apoptosis via the regulation of pro-apoptotic and anti-apoptotic proteins in pancreatic adenocarcinoma BxPC-3 cells by Chinese medicinal herbs Scutellaria barbata and Oldenlandia diffusa

Abstract Apoptosis is a controlled program cell death process that the body uses to combat cancer development. Pancreatic cancer is a devastating disease with a poor prognosis. It is usually symptom-free during development and is advanced at diagnosis. BxPC-3 is a pancreatic adenocarcinoma cell derived from a 61-year-old female with adenocarcinoma. Scutellaria barbata (SB) and Oldenlanda diffusa (OD) have been used in traditional Chinese medicine for treating liver, lung, and rectal cancers. They are included in most herbal cancer treatment formulas in Taiwan hospitals. We previously demonstrated that aqueous extracts of SB and OD selectively induced apoptosis in colon HCT 116 cancer cells but not in colon CCD 841 CoN normal epithelial cells by modulating the pro-apoptotic and anti-apoptotic proteins. They also induced and regulated apoptosis in pancreatic ductal carcinoma PANC-1 Cells. This study used the green/red/blue fluorescent Apoptosis/Necrosis Detection Kit and the Human Apoptosis Antibody Array - Membrane [43 Targets] Test (Abcam). Our fluorescent apoptosis data showed that a 2-hour treatment with 1.5 mg/mL and 3.0 mg/mL aqueous extract of SB and OD induced a statistically significant percentage of apoptosis in BxPC-3 cells (SB: 42.5 ± 3.5%, 48.5 ± 0.5% > 14.0% ± 4.0%, p < 0.05; OD: 72.0 ± 2.0%, 37.5 ± 1.5%> 14.0% ± 4.0%, p < 0.05) as compared to the negative control respectively. Similar results were obtained with the 4-hour incubation period (SB: 46.0 ± 8.0%, 54.5 ± 2.5% > 9.5% ± 2.5%, p < 0.05; OD: 49.0 ± 7.0%, 37.0 ± 0.0%> 9.5% ± 2.5%, p < 0.05). From the antibody array-membrane experiments, significant up-regulation of various pro-apoptotic proteins such as Bid, Bim, Bad, Bax, C3, C8, CD40, HTRA, IGFBP-4, IGFBP-4, p53, TNF-α, and TRAIL-2 as compared to the negative controls were observed; and regulation of anti-apoptotic proteins HSP27, IGF-II, and Survivin were also revealed. These results suggest that SB and OD contain phytochemicals that induce apoptosis in pancreatic adenocarcinoma BxPC-3 cells by regulating various apoptotic protein productions. Further study of their specific modulation effects of apoptosis is warranted to reveal their potential chemopreventive and therapeutic properties against pancreatic and other cancers. Citation Format: Christine Choi, Sarah Wolf, Rekha Isaac, Elim Choi, Abigail Shim, Angel Campana, Padma T. Uppala, Ryan Hayes, Brian Yuen Yau Wong. Induction of apoptosis via the regulation of pro-apoptotic and anti-apoptotic proteins in pancreatic adenocarcinoma BxPC-3 cells by Chinese medicinal herbs Scutellaria barbata and Oldenlandia diffusa [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7308.

PABPC1 promotes cell proliferation and metastasis in pancreatic adenocarcinoma by regulating COL12A1 expression.

The expression of cytoplasmic poly (A) binding protein-1 (PABPC1) has been reported in multiple cancer types. This protein is known to modulate cancer progression. However, the effects of PABPC1 expression in pancreatic adenocarcinoma (PAAD) have not been investigated. Here, we investigate the regulatory targets and molecular mechanisms of PABPC1 in PAAD. PABPC1 and collagen type XII α1 chain (COL12A1) expression in PAAD and their role in tumor prognosis and tumor stage were investigated using The Cancer Genome Atlasdatabase analysis. After silencing PABPC1, messenger RNA sequencing and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. The expression of differentially expressed genes (DEGs), cell viability, apoptosis, and cell migration and invasion were explored using reverse transcription-quantitative polymerase chain reaction, Cell Counting Kit-8assay, flow cytometry assay, and transwell assay, respectively. The relationship between PABPC1 and COL12A1 expression was assessed by Pearson's correlation analysis. The regulatory function of COL12A1 in PABPC1-affected BXPC3 cell behavior was studied after COL12A1 was overexpressed. PABPC1 and COL12A1 expression was upregulated in patients with PAAD and was linked to poor prognosis. Four hundred and seventy-four DEGs were observed in BXPC3 cells after PABPC1 silencing. GO and KEGG analyses revealed that the top 10 DEGs were enriched in cell adhesion pathways. Additionally, PABPC1 silencing inhibited cell viability, migration, and invasion and accelerated apoptosis in BXPC3 cells. PABPC1 silencing increased AZGP1 and ARHGAP30 expression and decreased CAV1 and COL12A1 expression in BXPC3 cells. PABPC1 positively mediated COL12A1 expression, whereas PABPC1 knockdown induced the inhibition of BXPC3 cell proliferation, migration, and invasion. The results of this study indicate that PABPC1 may function as a tumor promoter in PAAD, accelerating BXPC3 cell proliferation and metastasis by regulating COL12A1 expression.

Rational design and development of novel NAE inhibitors for the treatment of pancreatic cancer

Pancreatic cancer remains clinically challenging because of the lack of efficient targeted therapies and high aggressiveness. NEDD8 activating enzyme (NAE) plays a critical role in various cellular functions in cancers. Herein, we report the synthesis, optimization, and evaluation of a new series of pyrido[2,3-d]pyrimidin-7(8H)-one derivative as highly selective and efficacious NAE inhibitors, enabling rapid degradation of related substrates and potent inhibition of BxPC-3 cell proliferation. Moreover, western blot assays demonstrated that compound 51 could inhibit NAE activity, resulting in apoptosis in BxPC-3 cells. Furthermore, compound 51 induced cell apoptosis in vitro and inhibited tumor growth in BxPC-3 xenograft models. Our work established that 51 was a highly potent and efficacious NAE inhibitor, representing an effective strategy and great potential as a new targeted therapy for pancreatic cancer. Graphical abstract

Design, Synthesis and Biological Evaluation of Coumarin Derivatives as NEDD8 Activating Enzyme Inhibitors in Pancreatic Cancer Cells.

NEDD8 (neural precursor cell expressed developmentally downregulated protein 8) is one of the ubiquitin-like proteins which is activated by the NEDD8 activating enzyme (NAE). The overexpressed NAE can cause a variety of diseases such as numerous cancer types and inflammatory diseases. The selective inhibition of NAE could mediate the rate of ubiquitination and the subsequent degradation of proteins associated with cancer so as to achieve the purpose of treatment. In this article, we decided to study the synthesis and screening of coumarin scaffold derivatives against cancer cell lines, specifically the human pancreatic cancer cell line BxPC-3. Twenty-four targeted compounds were synthesized, and their anti-proliferative activity against three cancer cell lines, cytotoxicity against three normal cell lines through CCK-8 and MTT assay were evaluated to screen out the candidate compound. Then the target was further confirmed by both enzyme and cell-based experiments, as well as cell apoptosis research. Several new 4-position substituted coumarin derivatives (12a~x) were synthesized and most of them exhibit antiproliferative activity in three cancer cell lines. A series of experiments were performed to identify the best candidate compound 12v. This compound displayed the highest potency against BxPC-3 with an IC50 value of 0.28 μM. It can also inhibit NAE activity in enzyme and cellbased assay, and induce CRLs-mediated accumulation of the substrate and apoptosis in BxPC-3 cells. Meanwhile, it exhibited relatively low toxicity in three normal cells. Based on these results, we found that compound 12v inhibited NAE activity in enzyme and cell-based systems and induced apoptosis in BxPC-3 cells. Additionally, it also had a low toxicity. These results suggested that 12v may be promising lead compounds for the development of new anticancer drugs.

Hydroxychloroquine Promotes Bcl-xL Inhibition-induced Apoptosis in BxPC-3 Human Pancreatic Cancer Cells.

Anti-apoptotic proteins, including Bcl-2 and Bcl-xL, hinder cancer treatment, and several drugs targeting these molecules have been developed. One is ABT-263 (navitoclax), which targets Bcl-2, Bcl-xL, and Bcl-w. On the other hand, hydroxychloroquine (HCQ) has been used as a drug for malaria infection and autoimmune disease. HCQ can exert a similar effect as chloroquine with fewer adverse events. In addition, HCQ exerts antitumor activity. In the present study, the effects of HCQ on ABT-263-induced antitumor activities were examined using three human pancreatic cancer cell lines (PANC-1, MiaPaCa-2, and BxPC-3). In vitro effects of HCQ and ABT-263 were examined by cell viability, colony-forming assays, and flow cytometry. Protein expression was determined by immunoblotting. In vivo effects of HCQ and ABT-263 were examined by a xenograft mice model. Combined treatment with HCQ and ABT-263 synergistically decreased the viability of only BxPC-3 cells. This synergistic effect was not observed when HCQ was combined with ABT-199, an inhibitor specific to Bcl-2. The combination of HCQ and ABT-263 induced caspase-dependent apoptosis. Protein expression of Bcl-xL was more highly expressed in BxPC-3 cells than in the other two cell lines, and the combination of HCQ with a Bcl-xL inhibitor or siRNA-mediated knockdown of Bcl-xL induced apoptosis in BxPC-3 cells. Combination therapy with HCQ and ABT 737, an ABT-263 analogue, suppressed the in vivo growth of BxPC-3 with transient body-weight loss. HCQ effectively promotes Bcl-xL inhibition-induced apoptosis in BxPC-3 human pancreatic cancer cells.

Generation of a poly-functionalized indolizine scaffold and its anticancer activity in pancreatic cancer cells

A highly efficient domino [4 + 2] annulation process was employed to construct a novel indolizine chemical scaffold. Biological investigation led us to identify 6w as a potent anticancer agent. 6w significantly inhibited cell viability in BxPC3 pancreatic cancer, MCF7 breast cancer, and PC3 prostate cancer cell lines with IC50 values of 0.47 ± 0.04, 1.82 ± 0.08 and 2.68 ± 0.08 µM, respectively. Remarkably, 6w showed a weak effect on cell viability of nontumorigenic human keratinocyte cell line HaCaT compared to the above three types of cancer cells. 6w most potently inhibited cell viability of BxPC3 cells, and 6w also potently reduced cell migration and induced apoptosis in BxPC3 cells through activation of caspase-3 and cleavage of PARP in a dose-dependent manner. These results suggest that 6w can be used for the development of potential anticancer drugs for the treatment of pancreatic cancer.

Cardamonin Induces Apoptosis and Suppresses the Proliferation of BxPC3 Cells

Pancreatic cancer is one of the most lethal solid malignancies featured by rapid disease progression with no significant clinical symptoms and the treatment of pancreatic cancer remains unmet clinical need. Thus, to find a novel and potent therapeutic strategy for the treatment of pancreatic cancer is highly desired. The present study investigated the role and molecular mechanisms of cardamonin in pancreatic cancer cell line BxPC3. Cells are treated with cardamonin for 48 h and 72 h and evaluated cytotoxicity of cardamonin by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Clonogenic assay was applied to observe the anti-proliferation of cardamonin in pancreatic cancer cell line BxPC3 cells. Hoechst staining and flow cytometry were used to verify the mechanism of anti-proliferation of cardamonin in pancreatic cancer cell line BxPC3 cells. The expression of apoptosis-related proteins was evaluated by western blotting to investigate the molecular mechanism of cardamonin in pancreatic cancer cell line BxPC3 cells. The results of the present study showed that cardamonin inhibited the proliferation of pancreatic cancer cell line BxPC3 cells with the half maximal inhibitory concentration value of 28.86 μM and 22.83 μM at 48 h and 72 h respectively. Moreover, cardamonin induced apoptosis in BxPC3 cells via deoxyribonucleic acid damage to inhibit the pancreatic cancer. Overall, these results suggested that cardamonin has the potential to be used as a new therapeutic approach for pancreatic cancer.

Design, Synthesis, and Biological Evaluation of 4-amino Substituted 2Hchromen- 2-one Derivatives as an NEDD8 Activating Enzyme Inhibitor in Pancreatic Cancer Cells.

NEDD8 activating enzyme (NAE) plays a critical role in various cellular functions in carcinomas. The selective inhibition of NAE could mediate the rate of ubiquitination and the subsequent degradation of proteins associated with cancer so as to achieve the purpose of treatment. In this article, we decided to study the synthesis and screening of 4-amino substituted 2H-chromen-2-one derivatives against cancer cell lines, specifically the human pancreatic cancer cell line BxPC-3. After synthesis of twenty targeted compounds, we evaluated their anti-proliferative activity against six cancer cell lines, cytotoxicity against three normal cell lines through MTT assay, and hemolysis to screen out the candidate compound, which was further conducted drug-like physical property measurement, target confirmation by enzyme-based experiment, cell apoptosis, and synergistic effect research. Starting from intermediates 4 and 5, several new 4-amino substituted 2H-chromen-2-one derivatives (9-28) were synthesized and evaluated for their cell activities using six cancer cell lines. We performed tests of cytotoxicity, hemolysis, ATP-dependent NAE inhibition in the enzyme- based system, apoptosis, and synergistic effect in BxPC-3 cells against the best candidate compound 21. Based on these results, we found that compound 21 inhibited NAE activity in an ATP-dependent manner in the enzyme-based system, induced apoptosis in BxPC-3 cells, and synergized with bortezomib on BxPC-3 cell growth inhibition. Additionally, it had low toxicity with reasonable Log P-value and water solubility.

Exploiting the 7-methylimidazo[1,5-a]pyrazin-8(7H)-one scaffold for the development of novel chemical inhibitors for Bromodomain and Extraterminal Domain (BET) family

The bromodomain and extraterminal (BET) family of proteins play a crucial role in promoting gene expression of critical oncogenes. Novel BET bromodomain inhibitors with excellent potency, drug metabolism and pharmacokinetics (DMPK) properties were in strong need for development. We reported a series of potential BET inhibitors through incorporation of imidazole into pyridine scaffold. Among them, a novel BET inhibitor with 7-methylimidazo[1,5-a]pyrazin-8(7H)-one core, compound 28, was considered to be the most promising for in-depth study. Compound 28 exhibited excellent BRD4-inhibitory activity with IC50 value of 33 nM and anti-proliferation potency with IC50 value of 110 nM in HL-60 (human promyelocytic leukemia) cancer cell lines. Western Blot indicated that compound 28 can effectively trigger apoptosis in BxPc3 cells by modulating the intrinsic apoptotic pathway. In conclusion, these results suggested that compound 28 has merely potential for leukemia treatment.

Nano-chemotherapy using cationic liposome that strategically targets the cell membrane potential of pancreatic cancer cells with negative charge

Negatively charged phosphatidylserine (PS) and sialic acid-containing glycosphingolipids (GM1) were observed to be over represented on the cell membranes of pancreatic cancer cells (BxPC-3) as opposed to normal pancreatic cells. Cationic liposomes (CL) were also found to selectively accumulate into the negatively charged cell membranes of BxPC-3 cells and inhibited their growth but have no effect on the viability of normal pancreatic cells. CL induced apoptosis in BxPC-3 cells via activation of caspase-3, -8, and -9 and mitochondrial events and inhibited tumor enlargement in xenograft mouse models of pancreatic cancer.

A novel small-molecule YLT256 inhibits proliferation and induces apoptosis both in vitro and in vivo in solid tumors

Pancreatic carcinoma is a still unsolved health problem all over the world with poor prognosis and high mortality rate. YLT256, a novel synthesized chemical small inhibitor, displays potent antineoplastic activities via inducing apoptosis both in vitro and in vivo. In this study, we found that YLT256 showed growth inhibition against a broad spectrum of human cancer cell lines and pancreatic cancer cell line BxPc-3 was the most sensitive with an IC50 of 0.42μM. We also found YLT256 could induce apoptosis of BxPc-3 cells in a dose-dependent manner. Western blot analysis revealed that the occurrence of its apoptosis was associated with activation of caspases-3 and -9, up-regulation of pro-apoptotic Bak, and down-regulation of anti-apoptotic Bcl-2. Moreover, YLT256-treated resulted in changes of mitochondrial membrane potential (Δψm), and generation of reactive oxygen species (ROS). Furthermore, our data also revealed that YLT256 suppressed the growth of established tumor-bearing xenograft models without obvious side effects. Immunohistochemical analyses and TUNEL assay revealed an increase in cleaved caspase-3-positive cells and TUNEL-positive cells, a decrease in Ki67-positive cells upon YLT256. Together, all the results of present study provided evidence demonstrating that YLT256 could be a promising potential drug candidate for pancreatic cancer therapy.

Anticancer effects on human pancreatic cancer cells of triterpenoids, polysaccharides and 1,3-β-D-glucan derived from the fruiting body of Antrodia camphorata.

Antrodia camphorata is a fungus native to Taiwan, and it is considered a precious medicinal agent. We analyzed triterpenoids, polysaccharides and 1,3-β-D-glucan, three major effective components in A. camphorata extracts (ACE). ACE exhibited a selective cytotoxic effect on BxPC-3 human pancreatic cancer cells. ACE markedly inhibited the migration ability of BxPC-3 cells. Treatment of BxPC-3 cells with ACE resulted in the increase of cells in the sub-G1 phase and G2/M phase arrest. Apoptosis was confirmed by validating phosphatidylserine externalization, the observation of characteristic chromatin condensation, and nuclear DNA fragmentation. ACE induced apoptosis in BxPC-3 cells through a mitochondria-dependent pathway by triggering an appropriate balance of bax/bcl-2, cytochrome c release, activation of caspase-9 and -3, and poly(ADP-ribose) polymerase cleavage. ACE shows great therapeutic potential due to its cytotoxic effects against BxPC-3 cells which include inhibiting cell migration and inducing mitochondria-mediated apoptosis.

Influence of microRNA-135a on proliferation and apoptosis of human pancreatic cancer cells

Objective To investigate the influence of miRNA-135a on the proliferation and apoptosis of the pancreatic cancer cells.Methods Methyl thiazol tetrazolium (MTT) was used to detect the inhibitory effect of 5-fluorouracil (5-Fu) on Bxpc-3 cells and the the appropriate concentration of 5-Fu was chosen in the following experiment.The apoptosis of Bxpc-3 cells was detected by using flow cytometry.Real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) was used to detect the mRNA expression level of miRNA-135a after the Bxpc-3 cells were treated with 5-Fu.The mimic and anti-miRNA oligonuclotide (AMO) were designed and transfected into Bxpc-3 cells,and the Bxpc-3 cells transfected with vehicle served as control group.FQ-PCR was used to detect the expression of miRNA-135a and the proliferation rate was detected by using MTT assay.Flow cytometry was used to detect the apoptosis of transfected Bxpc-3 cells after treatment with 5-Fu.Results The 5-Fu inhibited Bxpc-3 cells in a concentration-and time-dependent manner.With the increase of concentration and prolongation of time,the inhibition rate of 5-Fu was elevated.The miRNA-135a level was reduced to (0.45 ±0.02),(0.60 ±0.03)and (0.73 ± 0.02) times after treatment with 5-Fu (P ＜ 0.05 for all).After transfection the mRNA levels of miRNA-13a was elevated (9.73 ±0.83) times in the mimic groups and reduced to (0.26 ±0.01) times in the AMO groups as compared with the vehicle groups (P ＜ 0.05 for all).The proliferation of Bxpc-3 cells was elevated in the mimic group and reduced in the AMO group as compared with the vehicle group.The apoptosis of Bxpc-3 cells,which was (12.03 1.07) ％,was reduced significantantly in the mimic groups as compared with the vehicle groups [(20.61 ± 1.53) ％] (P ＜ 0.05),while the apoptosis was elevated in the AMO groups [(38.20 ±2.64)％] (P ＜0.05).Conclusion The level of miRNA-135a was lower in Bxpe-3 cells treated with 5-Fu.The up-regulated expression of miRNA-135a may promote the proliferation and reduce the apoptosis rate of Bxpc-3 cells.The down-regulated miRNA-135a may inhibit the proliferation and augment the apoptosis of Bxpc-3 cells. Key words: Pancreatic cancer; MicroRNA ; Apoptosis

In vitro and in vivo anticancer efficacy of silibinin against human pancreatic cancer BxPC-3 and PANC-1 cells

Silibinin suppresses the growth of many cancers; however, its efficacy against pancreatic cancer has not been evaluated in established preclinical models. Here, we investigated in vitro and in vivo effects of silibinin against lower and advanced stages of human pancreatic carcinoma cells. Silibinin (25–100μM) treatment for 24–72h caused a dose- and time-dependent cell growth inhibition of 27–77% (P<0.05–0.001) in BxPC-3 cells, and 22–45% (P<0.01–0.001) in PANC-1 cells. Silibinin showed a strong dose-dependent G1 arrest in BxPC-3 cells (upto 72% versus 45% in control; P<0.001), but a moderate response in advanced PANC-1 cells. Cell death observed in cell growth assay, was accompanied by up to 3-fold increase (P<0.001) in apoptosis in BxPC-3 cells, and showed only slight effect on PANC-1 cells. Dietary feeding of silibinin (0.5%, w/w in AIN-93M diet for 7weeks) inhibited BxPC-3 and PANC-1 tumor xenografts growth in nude mice without any apparent change in body weight gain and diet consumption. Tumor volume and weight were decreased by 47% and 34% (P⩽0.001) in BxPC-3 xenograft, respectively. PANC-1 xenograft showed slower growth kinetics and silibinin decreased tumor volume by 34% (P<0.001) by 7weeks. Another 4weeks of silibinin treatment to PANC-1 xenograft showed 28% and 33% decrease in tumor volume and weight, respectively. Silibinin-fed group of BxPC-3 tumors showed decreased cell proliferation and angiogenesis and an increased apoptosis, however, considerable inhibitory effect was observed only for angiogenesis in PANC-1 tumors. Overall, these findings show both in vitro as well as in vivo anticancer efficacy of silibinin against pancreatic cancer that could involve inhibition of cell proliferation, cell cycle arrest, apoptosis induction and/or decrease in tumor angiogenesis.

The Antitumor Activity of Recombinant Antitumor Antiviral Protein and the Associated Molecular Mechanism in Pancreatic Tumor Cells

Pancreatic cancer has poor prognosis and high mortality. Currently, the therapy of pancreatic cancer remains a challenge. In this study, we compared the antitumor activity of the recombinant antitumor antiviral protein (RAAP), an improved interferon, with gemcitabine, a classic chemotherapy agent used for pancreatic cancer treatment. The proliferation of Bx-PC3 pancreatic cancer cells was evaluated by an MTT assay. Cell cycle arrest and apoptosis were evaluated by flow cytometry and annexin V-FITC/propidium iodide double staining, respectively. The expressions of matrix metalloproteinase (MMP)-2, MMP-9, caspase-3, caspase-8, and caspase-9 genes were evaluated by reverse transcription-polymerase chain reaction and the Western blot analysis. A xenograft pancreatic cancer model was established by inoculating Bx-PC3 cells into athymic nude mice. The antitumor activity of RAAP and gemcitabine was tested in the xenograft tumor model. RAAP significantly inhibited proliferation, induced cell cycle arrest, and induced apoptosis in Bx-PC3 cells in vitro and delayed tumor growth in vivo. The antitumor activity of 20 ng/mL of RAAP was a little more effective than 10 μM of gemcitabine. The antitumor activity of RAAP was associated with its role in inducing caspase-3 and caspase-8 expression as well as downregulating MMP-2 expression. RAAP can effectively suppress human pancreatic cancer cell growth in vitro and in vivo. The antitumor efficacy of RAAP is similar to gemcitabine.

Abstract 2822: Targeting Mcl-1 regulated apoptosis pathway as a novel therapy in pancreatic cancer

Abstract Resistance of pancreatic cancer to current treatment protocols as a consequence of apoptosis defects is a major cause of treatment failure. Therapeutic approaches that specifically target components of these molecular mechanisms are urgently needed. The anti-apoptotic protein Mcl-1, a member of the Bcl-2 family proteins, has emerged as a promising therapeutic target. Consistent with its anti-apoptotic function, the overexpression of Mcl-1 in pancreatic cancer cells has been associated with tumor progression and resistance to current chemotherapeutics. Applying high throughput screening approach several promising lead compounds as selective Mcl-1 inhibitors were identified and evaluated. Based on one of the most promising lead compounds we have designed and synthesized several analogues. The most potent compound, UMI-77, binds to Mcl-1 protein with Ki = 205 nM determined by fluorescence polarization based assay and shows selectivity over Bcl-2 and Bcl-xL. NMR spectroscopy studies demonstrate that UMI-77 binds to the same BH3 domain of Mcl-1 as the Bim BH3 peptide and antagonizes Mcl-1 function. UMI-77 inhibits cell growth and induces apoptosis in pancreatic cancer cells with high Mcl-1 levels (BxPC-3 and Panc-1) in a time and dose-dependent manner, accompanied by caspase-3 activation. Applying siRNA approach, the transient suppression of Mcl-1 abrogated UMI-77 mediated apoptosis in BxPC-3 cells, demonstrating that UMI-77 as a single agent can inhibit cell growth and induce apoptosis in pancreatic cells in a Mcl-1 dependent manner. Co-immunoprecipitation experiments revealed that UMI-77 blocks the heterodimerization of Mcl-1/Bax and Mcl-1/Bak in cells. By using murine embryonic fibroblasts (MEFs), wild type and deficient in both Bax and Bak (double knock out), it was demonstrated that the cytotoxic activity and induction of apoptosis by UMI-77 mainly depend on Bax and/or Bak, suggesting that function as BH3 mimetic. In order to verify the clinical utility of UMI-77, we tested its in vivo efficacy using BxPC-3 xenograft model. The maximum tolerated dose (MTD) of UMI-77 in SCID mice was determined to be 60mg/kg i.v. We further demonstrated that UMI-77 at MTD showed statistically significant and effective tumor growth inhibition in BxPC-3 xenograft model. At the end of the treatment, western blot analysis for different molecular markers on lysates isolated from tumors harvested from SCID mice revealed enhancement of proapoptotic markers in UMI-77 treated animals compared to control. Collectively, these promising findings warrant further chemical modifications of this compound and optimization toward developing a new class of anticancer drugs, Mcl-1 inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2822. doi:10.1158/1538-7445.AM2011-2822

Downregulation of STAT3 signaling induces apoptosis but also promotes anti-apoptotic gene expression in human pancreatic cancer cell lines

Signal transducer and activator of transcription 3 (STAT3) is a key regulator of cytokine signaling pathways that regulates gene expression. In pancreatic cancer, constitutive activation of STAT3 contributes to oncogenesis by preventing apoptosis through upregulation of anti-apoptotic proteins. We have examined the inhibition of STAT3 as a potential therapeutic approach in pancreatic cancer. siRNA targeting STAT3 was used to evaluate the role of STAT3 in modulating the expression of Survivin/BIRC5 and BCL-xL in the pancreatic cancer cell lines PANC-1 and BxPC-3 and induction of apoptosis. Expression of STAT3, Survivin/BIRC5, and BCL-xL on mRNA and protein level was measured by real-time RT-PCR and Western blot analysis 24, 48, and 72 h after transfection. STAT3 downregulation resulted in a decrease of cell viability in both cell lines and induced apoptosis in BxPC-3 cells. Despite significant inhibition of STAT3, the expression of the anti-apoptotic genes Survivin/BIRC5 and BCL-xL were not subsequently downregulated. Even more, the cell line BxPC-3 shows a significant increase of Survivin/BIRC5 and BCL-xL mRNA after 48-72 h as a result of STAT3 downregulation. Inactivation of STAT3 in pancreatic cancer cell lines induces apoptosis but also may promote the expression of anti-apoptotic genes.

Influence of betulinic acid on proliferation, migration, cell cycle and apoptosis of pancreatic cancer cells

To investigate the effect of betulinic acid (BA) on the proliferation, migration, apoptosis and cell cycle of pancreatic cancer cells (BxPC-3) in vitro and elucidate the underlying. The effect of BA on the proliferation of BxPC-3 was measured by using sulforhodamine B (SRB) assay. Migratory ability of BxPC3 cells were detected by wound healing assay, and the morphological change was observed with light microscope. The influence of BA on cell cycle of BxPC-3 cells was tested by flow cytometry (FCM). Apoptosis was analyzed by using Hochest33342-PI double staining. Western blot technologies were applied to detect the expression of Bcl-2 and Bax. BA exhibited significant cell proliferation and migration inhibition, as well as its potency of inducing apoptosis in BxPC-3 cells in vitro in a dose-dependent manner. The IC50 value for 72 h was 16.54 mg x L(-1). Cell migration was significantly inhibited at 5 mg x L(-1) of BA. Cells treated with BA showed increased cell population in G0 phase, with decreased G2/M phase population. The expression of Bax and Bcl-2 was up and down-regulated respectively in BA-treated BxPC-3 cells in a dose-dependent manner. BA exerted potent effect on growth inhibition, G0 cell cycle arrest and induction of apoptosis in BxPC-3 cells in vitro, possibly associated with the down-regulation of Bcl-2 and up-regulation of Bax expression. The potent antitumor capacity of BA suggested that it could be a promising new anticancer agent in human pancreatic cancer treatment.

Benzyl isothiocyanate sensitizes human pancreatic cancer cells to radiation therapy

Increase in systemic toxicity and resistance are the major drawbacks of radiation therapy in the treatment of pancreatic cancer. We have shown previously that BITC inhibits the growth of human pancreatic cancer cells and induces apoptosis. Here we determined whether BITC could sensitize BxPC-3 cells and increase the therapeutic potential of gamma-irradiation. Cells were pretreated with 2.5 microM BITC for 24h followed by exposure to 5 Gy of gamma-irradiation and were allowed to grow for another 24 or 48 h before being analyzed. Combination of BITC and gamma-irradiation significantly reduced survival of cells and caused significantly enhanced arrest of cells in G2/M phase as compared to cells exposed to gamma-irradiation alone. G2/M arrest was associated with DNA damage leading to the phosphorylation of ATR (Ser-428), Chk2 (Thr-68), Cdc25C (Ser-216), Cdk-1 (Tyr-15) and induction of p21Waf1/Cip1. However, combination treatment after 48 h caused 2.8-fold increase in apoptosis in BxPC-3 cells. Apoptosis at 48 h was associated with NF-kappa B inhibition and p38 activation. Taken together, results of the present study suggest that the apoptosis-inducing effect of gamma-irradiation can be increased by BITC.

Sensitization of pancreatic cancer cells to ApoG2 induced killing by curcumin

13152 Background: In pancreatic cancer, several important survival molecules such as NF-kappa B and Bcl-2 or Bcl-XL are highly activated. Thus agents that inhibit NF-KappaB activation, together with agents that inhibit Bcl-2 or Bcl-XL protein function, may lead to enhanced cell killing. ApoG2, a new derivative of natural product gossypol, is a potent small molecule inhibitor of Bcl-2 and Bcl-XL proteins. Curcumin, an active ingredient from the rhizome of the plant, Curcuma longa, has anti-inflammatory and anti-cancer activities and works by inactivation of NF-Kappa B activity. In this study we have investigated the effects of curcumin on ApoG2 induced apoptosis. Methods: The BxPC-3 human pancreatic cancer cell line was used in this study. ApoG2 was dissolved in DMSO at 1 mM stock solution, and curcumin was dissolved in DMSO at 20 mM stock solution. Cell viability was determined by the standard MTT assay and apoptosis was measured by histone/DNA ELISA method. Results: Pretreatment of human pancreatic cell line BxPC-3 with curcumin sensitizes ApoG2 induced cell killing. In the dose escalation with ApoG2 at 48 hrs on MTT assay, 250 nM showed 75% cell viability, while 2.5 uM of curcumin at 72 hrs showed 80% cell viability. Pretreatment with curcumin for 24 hrs followed by ApoG2 at 48 hrs showed 55% cell viability (p = 0.0005). Apoptosis by histone/DNA ELISA showed 4-fold induction of cell death with ApoG2 at 48 hrs, 7-fold induction of cell death with curcumin at 72 hrs. However, pretreatment with curcumin for 24 hrs followed by ApoG2 at 48 hrs showed 10-fold induction in apoptosis compared to the control. Conclusions: From these results, we conclude that curcumin enhanced ApoG2 induced cell growth inhibition and apoptosis of BxPC-3 cells. No significant financial relationships to disclose.