Novel Pancreatic Cancer Cell Lines Derived from Genetically Engineered Mouse Models of Spontaneous Pancreatic Adenocarcinoma: Applications in Diagnosis and Therapy

Pancreatic cancer (PC) remains one of the most lethal human malignancies with poor prognosis. Despite all advances in preclinical research, there have not been significant translation of novel therapies into the clinics. The development of genetically engineered mouse (GEM) models that produce spontaneous pancreatic adenocarcinoma (PDAC) have increased our understanding of the pathogenesis of the disease. Although these PDAC mouse models are ideal for studying potential therapies and specific genetic mutations, there is a need for developing syngeneic cell lines from these models. In this study, we describe the successful establishment and characterization of three cell lines derived from two (PDAC) mouse models. The cell line UN-KC-6141 was derived from a pancreatic tumor of a Kras^G12D;Pdx1-Cre (KC) mouse at 50 weeks of age, whereas UN-KPC-960 and UN-KPC-961 cell lines were derived from pancreatic tumors of Kras^G12D;Trp53^R172H;Pdx1-Cre (KPC) mice at 17 weeks of age. The cancer mutations of these parent mice carried over to the daughter cell lines (i.e. Kras^G12D mutation was observed in all three cell lines while Trp53 mutation was observed only in KPC cell lines). The cell lines showed typical cobblestone epithelial morphology in culture, and unlike the previously established mouse PDAC cell line Panc02, expressed the ductal marker CK19. Furthermore, these cell lines expressed the epithelial-mesenchymal markers E-cadherin and N-cadherin, and also, Muc1 and Muc4 mucins. In addition, these cell lines were resistant to the chemotherapeutic drug Gemcitabine. Their implantation in vivo produced subcutaneous as well as tumors in the pancreas (orthotopic). The genetic mutations in these cell lines mimic the genetic compendium of human PDAC, which make them valuable models with a high potential of translational relevance for examining diagnostic markers and therapeutic drugs.     

Grape Proanthocyanidin Inhibit Pancreatic Cancer Cell Growth In Vitro and In Vivo through Induction of Apoptosis and by Targeting the PI3K/Akt Pathway

Pancreatic cancer is an aggressive malignancy that is frequently diagnosed at an advanced stage with poor prognosis. Here, we report the chemotherapeutic effects of bioactive proanthocyanidins from grape seeds (GSPs) as assessed using In Vitro and In Vivo models. Treatment of human pancreatic cancer cells (Miapaca-2, PANC-1 and AsPC-1) with GSPs In Vitro reduced cell viability and increased G2/M phase arrest of the cell cycle leading to induction of apoptosis in a dose- and time-dependent manner. The GSPs-induced apoptosis of pancreatic cancer cells was associated with a decrease in the levels of Bcl-2 and Bcl-xl and an increase in the levels of Bax and activated caspase-3. Treatment of Miapaca-2 and PANC-1 cells with GSPs also decreased the levels of phosphatidylinositol-3-kinase (PI3K) and phosphorylation of Akt at ser(473). siRNA knockdown of PI3K from pancreatic cancer cells also reduced the phosphorylation of Akt. Further, dietary administration of GSPs (0.5%, w/w) as a supplemented AIN76A control diet significantly inhibited the growth of Miapaca-2 pancreatic tumor xenografts grown subcutaneously in athymic nude mice, which was associated with: (i) inhibition of cell proliferation, (ii) induction of apoptosis of tumor cells, (iii) increased expression of Bax, reduced expression of anti-apoptotic proteins and activation of caspase-3-positive cells, and (iv) decreased expression of PI3K and p-Akt in tumor xenograft tissues. Together, these results suggest that GSPs may have a potential chemotherapeutic effect on pancreatic cancer cell growth.     

Loss of p53 Attenuates the Contribution of IL-6 Deletion on Suppressed Tumor Progression and Extended Survival in Kras-Driven Murine Lung Cancer

Interleukin-6 (IL-6) is involved in lung cancer tumorigenesis, tumor progression, metastasis, and drug resistance. Previous studies show that blockade of IL-6 signaling can inhibit tumor growth and increase drug sensitivity in mouse models. Clinical trials in non-small cell lung cancer (NSCLC) reveal that IL-6 targeted therapy relieves NSCLC-related anemia and cachexia, although other clinical effects require further study. We crossed IL-6 ^-/- mice with Kras ^G12D mutant mice, which develop lung tumors after activation of mutant Kras ^G12D, to investigate whether IL-6 inhibition contributes to tumor progression and survival time in vivo. Kras ^G12D; IL-6 ^-/- mice exhibited increased tumorigenesis, but slower tumor growth and longer survival, than Kras ^G12D mice. Further, in order to investigate whether IL-6 deletion contributes to suppression of lung cancer metastasis, we generated Kras ^G12D; p53 ^flox/flox; IL-6 ^-/- mice, which developed lung cancer with a trend for reduced metastases and longer survival than Kras ^G12D; p53 ^flox/flox mice. Tumors from Kras ^G12D; IL-6 ^-/- mice showed increased expression of TNFα and decreased expression of CCL-19, CCL-20 and phosphorylated STAT3 (pSTAT3) than Kras ^G12D mice; however, these changes were not present between tumors from Kras ^G12D; p53 ^flox/flox; IL-6 ^-/- and Kras ^G12D; p53 ^flox/flox mice. Upregulation of pSTAT3 and phosphorylated AKT (pAKT) were observed in Kras ^G12D tumors with p53 deletion. Taken together, these results indicate that IL-6 deletion accelerates tumorigenesis but delays tumor progression and prolongs survival time in a Kras-driven mouse model of lung cancer. However, these effects can be attenuated by p53 deletion.     

Resveratrol inhibits pancreatic cancer stem cell characteristics in human and KrasG12D transgenic mice by inhibiting pluripotency maintaining factors and epithelial-mesenchymal transition

Background

Cancer stem cells (CSCs) can proliferate and self-renew extensively due to their ability to express anti-apoptotic and drug resistant proteins, thus sustaining tumor growth. Therefore, the strategy to eradicate CSCs might have significant clinical implications. The objectives of this study were to examine the molecular mechanisms by which resveratrol inhibits stem cell characteristics of pancreatic CSCs derived from human primary tumors and Kras^G12D transgenic mice.

Methodology/Principal Findings

Human pancreatic CSCs (CD133⁺CD44⁺CD24⁺ESA⁺) are highly tumorigenic and form subcutaneous tumors in NOD/SCID mice. Human pancreatic CSCs expressing high levels of CD133, CD24, CD44, ESA, and aldehyde dehydrogenase also express significantly more Nanog, Oct-4, Notch1, MDR1 and ABCG2 than normal pancreatic tissues and primary pancreatic cancer cells. Similarly, CSCs from Kras^G12D mice express significantly higher levels of Nanog and Oct-4 than pancreatic tissues from Pdx-Cre mice. Resveratrol inhibits the growth (size and weight) and development (PanIN lesions) of pancreatic cancer in Kras^G12D mice. Resveratrol inhibits the self-renewal capacity of pancreatic CSCs derived from human primary tumors and Kras^G12D mice. Resveratrol induces apoptosis by activating capase-3/7 and inhibiting the expression of Bcl-2 and XIAP in human CSCs. Resveratrol inhibits pluripotency maintaining factors (Nanog, Sox-2, c-Myc and Oct-4) and drug resistance gene ABCG2 in CSCs. Inhibition of Nanog by shRNA enhances the inhibitory effects of resveratrol on self-renewal capacity of CSCs. Finally, resveratrol inhibits CSC''s migration and invasion and markers of epithelial-mesenchymal transition (Zeb-1, Slug and Snail).

Conclusions/Significance

These data suggest that resveratrol inhibits pancreatic cancer stem cell characteristics in human and Kras^G12D transgenic mice by inhibiting pluripotency maintaining factors and epithelial-mesenchymal transition. In conclusion, resveratrol can be used for the management of pancreatic cancer.     

Evaluation of Efficacy of Radioimmunotherapy with 90Y-Labeled Fully Human Anti-Transferrin Receptor Monoclonal Antibody in Pancreatic Cancer Mouse Models

Objective

Pancreatic cancer is an aggressive tumor and the prognosis remains poor. Therefore, development of more effective therapy is needed. We previously reported that ⁸⁹Zr-labeled TSP-A01, an antibody against transferrin receptor (TfR), is highly accumulated in a pancreatic cancer xenograft, but not in major normal organs. In the present study, we evaluated the efficacy of radioimmunotherapy (RIT) with ⁹⁰Y-TSP-A01 in pancreatic cancer mouse models.

Methods

TfR expression in pancreatic cancer cell lines (AsPC-1, BxPC-3, MIAPaCa-2) was evaluated by immunofluorescence staining. ¹¹¹In-labeled anti-TfR antibodies (TSP-A01, TSP-A02) were evaluated in vitro by cell binding assay with the three cell lines and by competitive inhibition assay with MIAPaCa-2. In vivo biodistribution was evaluated in mice bearing BxPC-3 and MIAPaCa-2 xenografts. Tumor volumes of BxPC-3 and MIAPaCa-2 were sequentially measured after ⁹⁰Y-TSP-A01 injection and histological analysis of tumors was conducted.

Results

MIAPaCa-2 cells showed the highest TfR expression, followed by AsPC-1 and BxPC-3 cells. ¹¹¹In-TSP-A01 and ¹¹¹In-TSP-A02 bound specifically to the three cell lines according to TfR expression. The dissociation constants for TSP-A01, DOTA-TSP-A01, TSP-A02, and DOTA-TSP-A02 were 0.22, 0.28, 0.17, and 0.22 nM, respectively. ¹¹¹In-TSP-A01 was highly accumulated in tumors, especially in MIAPaCa-2, but this was not true of ¹¹¹In-TSP-A02. The absorbed dose for ⁹⁰Y-TSP-A01 was estimated to be 8.3 Gy/MBq to BxPC-3 and 12.4 Gy/MBq to MIAPaCa-2. MIAPaCa-2 tumors treated with 3.7 MBq of ⁹⁰Y-TSP-A01 had almost completely disappeared around 3 weeks after injection and regrowth was not observed. Growth of BxPC-3 tumors was inhibited by 3.7 MBq of ⁹⁰Y-TSP-A01, but the tumor size was not reduced.

Conclusion

⁹⁰Y-TSP-A01 treatment achieved an almost complete response in MIAPaCa-2 tumors, whereas it merely inhibited the growth of BxPC-3 tumors. ⁹⁰Y-TSP-A01 is a promising RIT agent for pancreatic cancer, although further investigation is necessary to improve the efficacy for the radioresistant types like BxPC-3.     

Anti-tumor activity of the X-linked inhibitor of apoptosis (XIAP) inhibitor embelin in gastric cancer cells

This study investigated the anticancer effects of embelin in human gastric cancer cells and the underlying molecular mechanisms. Gastric cancer cells were treated with embelin and 5-FU for methyl-thiazolyl-tetrazolium bromide cell viability assay and flow cytometric detection of cell viability and apoptosis. Protein pathway array (PPA) and Western blot were used to investigate differentially expressed proteins in embelin-treated gastric cancer cells. Embelin reduced gastric cancer cell viability, induced apoptosis, and enhanced 5-FU antitumor activity in gastric cancer cells. Mechanistically, embelin induced cell cycle arrest at the S and G2/M phases. Molecularly, embelin downregulated expression of X-linked inhibitor of apoptosis and cell cycle-regulatory proteins, such as CDK1, CDC25B, CDC25C, cyclinB1, and CDK2. PPA analysis showed that embelin modulated several pathways that are associated with cell growth and apoptosis, such as PI3K/AKT, JAK/STAT, p38 MAPK, and p53. The data from the current study implied that reduction of gastric cancer cell viability after treatment with embelin was through cell cycle arrest at the S and G2/M phases and apoptosis.     

Oncogenic Kras Initiates Leukemia in Hematopoietic Stem Cells

How oncogenes modulate the self-renewal properties of cancer-initiating cells is incompletely understood. Activating KRAS and NRAS mutations are among the most common oncogenic lesions detected in human cancer, and occur in myeloproliferative disorders (MPDs) and leukemias. We investigated the effects of expressing oncogenic Kras^G12D from its endogenous locus on the proliferation and tumor-initiating properties of murine hematopoietic stem and progenitor cells. MPD could be initiated by Kras^G12D expression in a highly restricted population enriched for hematopoietic stem cells (HSCs), but not in common myeloid progenitors. Kras^G12D HSCs demonstrated a marked in vivo competitive advantage over wild-type cells. Kras^G12D expression also increased the fraction of proliferating HSCs and reduced the overall size of this compartment. Transplanted Kras^G12D HSCs efficiently initiated acute T-lineage leukemia/lymphoma, which was associated with secondary Notch1 mutations in thymocytes. We conclude that MPD-initiating activity is restricted to the HSC compartment in Kras^G12D mice, and that distinct self-renewing populations with cooperating mutations emerge during cancer progression.     

Targeting Cancer-Related Inflammation: Chinese Herbal Medicine Inhibits Epithelial-to-Mesenchymal Transition in Pancreatic Cancer

Pancreatic cancer is an almost universally fatal disease resulting from early invasion of adjacent structures and metastasis and the lack of an effective treatment modality. Our previous studies have shown that Qingyihuaji Formula (QYHJ), a seven-herb Chinese medicine formula, had significant anti-cancer effects in pancreatic cancer. Here, we examined the effects of QYHJ on pancreatic cancer cell invasion and metastasis and the potential associated mechanism(s). We found that QYHJ inhibited both tumor growth and metastasis in nude mice with human pancreatic cancer cell xenografts. Further study indicated that QYHJ inhibited epithelial-to-mesenchymal transition (EMT), which is characterized by increased E-cadherin expression and decreased vimentin, N-cadherin and Slug expression. Interleukin 6 (IL-6), a pro-inflammatory cytokine produced mainly by macrophages, could promote cancer cell EMT and invasion. In contrast, treatment with QYHJ inhibited cancer-related inflammation in tumors by decreasing infiltration of tumor-associated macrophages and IL-6 production, thus preventing cell invasion and metastasis. These results suggested that the Chinese herbal medicine QYHJ could inhibit pancreatic cancer cell invasion and metastasis in part by reversing tumor-supporting inflammation.     

PPDPF Promotes the Progression and acts as an Antiapoptotic Protein in Non-Small Cell Lung Cancer

Resistance to radiotherapy is frequently observed in the clinic and leads to poor prognosis of non-small cell lung cancer (NSCLC). How to overcome resistance to radiotherapy is a challenge in the treatment of NSCLC. In this study, PPDPF was found to be upregulated in NSCLC tissues and cell lines, and its expression negatively correlated with the overall survival of patients with NSCLC. PPDPF promoted the growth, colony formation and invasion of lung cancer cells. Moreover, knockout of PPDPF inhibited tumorigenesis in the KL (Kras^G12D; LKB1^f/f) mouse model of lung cancer. Additionally, overexpression of PPDPF led to radioresistance in lung cancer cells, and knockdown of PPDPF sensitized lung cancer cells to radiotherapy. Mechanistically, PPDPF interacted with BABAM2 (an antiapoptotic protein) and blocked its ubiquitination by MDM2, thus stabilizing BABAM2 and promoting the radioresistance of lung cancer cells. Our present study suggested PPDPF as a therapeutic target in NSCLC.     

Anti-Tumour Efficacy of Capecitabine in a Genetically Engineered Mouse Model of Pancreatic Cancer

Capecitabine (CAP) is a 5-FU pro-drug approved for the treatment of several cancers and it is used in combination with gemcitabine (GEM) in the treatment of patients with pancreatic adenocarcinoma (PDAC). However, limited pre-clinical data of the effects of CAP in PDAC are available to support the use of the GEMCAP combination in clinic. Therefore, we investigated the pharmacokinetics and the efficacy of CAP as a single agent first and then in combination with GEM to assess the utility of the GEMCAP therapy in clinic. Using a model of spontaneous PDAC occurring in Kras^G12D; p53^R172H; Pdx1-Cre (KPC) mice and subcutaneous allografts of a KPC PDAC-derived cell line (K8484), we showed that CAP achieved tumour concentrations (∼25 µM) of 5-FU in both models, as a single agent, and induced survival similar to GEM in KPC mice, suggesting similar efficacy. In vitro studies performed in K8484 cells as well as in human pancreatic cell lines showed an additive effect of the GEMCAP combination however, it increased toxicity in vivo and no benefit of a tolerable GEMCAP combination was identified in the allograft model when compared to GEM alone. Our work provides pre-clinical evidence of 5-FU delivery to tumours and anti-tumour efficacy following oral CAP administration that was similar to effects of GEM. Nevertheless, the GEMCAP combination does not improve the therapeutic index compared to GEM alone. These data suggest that CAP could be considered as an alternative to GEM in future, rationally designed, combination treatment strategies for advanced pancreatic cancer.     

Bmi1 Enhances Tumorigenicity and Cancer Stem Cell Function in Pancreatic Adenocarcinoma

Background

Bmi1 is an integral component of the Polycomb Repressive Complex 1 (PRC1) and is involved in the pathogenesis of multiple cancers. It also plays a key role in the functioning of endogenous stem cells and cancer stem cells. Previous work implicated a role for cancer stem cells in the pathogenesis of pancreatic cancer. We hypothesized that Bmi1 plays an integral role in enhancing pancreatic tumorigenicity and the function of cancer stem cells in pancreatic ductal adenocarcinoma.

Methods

We measured endogenous Bmi1 levels in primary human pancreatic ductal adenocarcinomas, pancreatic intraepithelial neoplasias (PanINs) and normal pancreas by immunohistochemistry and Western blotting. The function of Bmi1 in pancreatic cancer was assessed by alteration of Bmi1 expression in several cell model systems by measuring cell proliferation, cell apoptosis, in vitro invasion, chemotherapy resistance, and in vivo growth and metastasis in an orthotopic model of pancreatic cancer. We also assessed the cancer stem cell frequency, tumorsphere formation, and in vivo growth of human pancreatic cancer xenografts after Bmi1 silencing.

Results

Bmi1 was overexpressed in human PanINs, pancreatic cancers, and in several pancreatic cancer cell lines. Overexpression of Bmi1 in MiaPaCa2 cells resulted in increased proliferation, in vitro invasion, larger in vivo tumors, more metastases, and gemcitabine resistance while opposite results were seen when Bmi1 was silenced in Panc-1 cells. Bmi1 was overexpressed in the cancer stem cell compartment of primary human pancreatic cancer xenografts. Pancreatic tumorspheres also demonstrated high levels of Bmi1. Silencing of Bmi1 inhibited secondary and tertiary tumorsphere formation, decreased primary pancreatic xenograft growth, and lowered the proportion of cancer stem cells in the xenograft tissue.

Conclusions

Our results implicate Bmi1 in the invasiveness and growth of pancreatic cancer and demonstrate its key role in the regulation of pancreatic cancer stem cells.     

Long non-coding RNA OIP5-AS1 promotes pancreatic cancer cell growth through sponging miR-342-3p via AKT/ERK signaling pathway

Opa-interacting protein 5 antisense RNA 1 (OIP5-AS1), a long non-coding RNA (lncRNA), has been reported to link with the progression of some cancers. However, its biological functions and underlying molecular mechanisms in pancreatic cancer are largely unknown. The aim of this study was to investigate the role of lncRNA OIP5-AS1 in pancreatic cancer. Quantitative real-time PCR analysis revealed that OIP5-AS1 is highly expressed in pancreatic cancer tissues versus adjacent non-tumor tissues. In vitro functional assays showed that downregulation of OIP5-AS1 or overexpression of miR-342-3p inhibited the proliferation, decreased Ki67 expression, and induced cell cycle arrest in pancreatic cancer cells. The expression of cyclinD1, CDK4, and CDK6 was decreased by knockdown of OIP5-AS1. Moreover, we found that OIP5-AS1 acted as a miR-342-3p sponge to suppress its expression and function. Dual-luciferase assay confirmed the interaction of OIP5-AS1 and miR-342-3p and verified anterior gradient 2 (AGR2) as a direct target of miR-342-3p. Results showed that depletion of miR-342-3p abolished the inhibitory effects of OIP5-AS1 knockdown on pancreatic cancer cell growth. The expression of Ki67, AGR2, cyclinD1, CDK4, CDK6, p-AKT, and p-ERK1/2 was reversed by silencing of miR-342-3p in pancreatic cancer cells with OIP5-AS1 knockdown. Further, knockdown of OIP5-AS1 suppressed tumor growth in a xenograft mouse model of pancreatic cancer. OIP5-AS1 induced pancreatic cancer progression via activation of AKT and ERK signaling pathways. Therefore, we demonstrate that OIP5-AS1 functions as oncogene in pancreatic cancer and its downregulation inhibits pancreatic cancer growth by sponging miR-342-3p via targeting AGR2 through inhibiting AKT/ERK signaling pathway.

     

虎杖提取物对人胰腺癌细胞系Panc-1增殖与凋亡的影响

目的:通过虎杖提取物干预人胰腺癌细胞系Panc-1,探讨虎杖提取物对人胰腺癌细胞增殖凋亡表型的影响。方法:制备不同浓度(0、10、50、100、150、200μg/m L)的虎杖提取物,将各个浓度的虎杖提取物分别加入待处理的人胰腺癌Panc-1细胞系中持续培养24 h后,利用CCK-8(cell counting kit-8)法检测细胞株Panc-1的增殖活性;将100μg/m L虎杖提取物处理人胰腺癌细胞系Panc-124 h后,利用流式细胞术(FCM)检测其细胞周期及凋亡分布;100μg/m L虎杖提取物处理人胰腺癌细胞株Panc-124 h后,提取细胞总RNA及总蛋白,后续利用实时荧光定量PCR及Western blot分别检测人胰腺癌细胞株Panc-1增殖标志基因PCNA、CDK2及凋亡标志基因BAD、BAX的转录和翻译水平。结果:CCK-8结果表明虎杖提取物对人胰腺癌细胞系Panc-1细胞增殖的抑制率随浓度增加;流式细胞术结果显示虎杖提取物抑制人胰腺癌细胞增殖促进其凋亡;荧光定量PCR和Western blot结果显示虎杖提取物能使人胰腺癌细胞增殖标志基因PCNA,CDK2表达量下降,凋亡标志基因BAD,BAX表达量上升。结论:虎杖提取物能够抑制人胰腺癌细胞系Panc-1细胞增殖并促进其凋亡。     

人胰腺癌细胞再增殖体外模型的建立

目的:细胞再增殖是导致胰腺癌放化疗失败的主要原因之一,但缺乏合适的用于研究胰腺癌再增殖的细胞模型。本研究拟建立简便、实用的胰腺癌细胞再增殖体外模型。方法:表达绿色荧光蛋白-荧光素酶(GFP-Luc)的慢病毒感染人胰腺癌细胞,经嘌呤霉素筛选,用荧光显微镜和流式细胞仪观察双标记细胞GFP表达情况,用生物成像检测双标记细胞Luc活性及分析细胞数量与Luc活性之间的关系。以X-线照射胰腺癌细胞制备饲养细胞,以相应的双标记肿瘤细胞为报告细胞进行共培养。对共培养细胞进行荧光显微镜观察和生物成像,以判断饲养细胞对报告细胞的生长促进作用。结果:通过表达GFP-Luc的慢病毒感染获得双标记人胰腺癌细胞,经荧光显微术、流式细胞术和生物成像术证实这些双标记的人胰腺癌细胞能有效地表达GFP和Luc活性,可作为报告细胞用于建立人胰腺癌再增殖细胞模型。将经X-线照射的饲养细胞和相应的报告细胞共培养,经荧光显微镜观察和生物成像分析,结果显示X-线照射过的饲养细胞对报告细胞的生长具有显著的促进作用。结论:成功建立了简便、实用的人胰腺癌再增殖体外模型,该模型能很好地模拟人体内胰腺癌细胞再增殖过程,为进一步研究胰腺癌细胞再增殖的分子机制提供了新的技术手段。     

In vitro and in vivo induction of apoptosis by capsaicin in pancreatic cancer cells is mediated through ROS generation and mitochondrial death pathway

Pancreatic cancer is one of the most common invasive malignancies and the fourth leading cause of cancer related mortality in U.S., thus developing new strategies to control pancreatic cancer is an important mission. We investigated the mechanism of capsaicin, the major pungent ingredient of red-chili pepper, in inducing apoptosis in pancreatic cancer cells. Treatment of AsPC-1 and BxPC-3 cells with capsaicin resulted in a dose-dependent inhibition of cell-viability and induction of apoptosis which was associated with the generation of ROS and persistent disruption of mitochondrial membrane potential. These effects were significantly blocked when the cells were pretreated with a general antioxidant N-acetyl cysteine (NAC). Exposure of AsPC-1 and BxPC-3 cells to capsaicin was also associated with increased expression of Bax, down-regulation of bcl-2, survivin and significant release of cytochrome c and AIF in the cytosol. On the contrary, above-mentioned effects were not observed in the normal acinar cells in response to capsaicin-treatment. Capsaicin-treatment resulted in the activation of JNK and JNK inhibitor SP600125 afforded protection against capsaicin-induced apoptosis. Furthermore, capsaicin when given orally markedly suppressed the growth of AsPC-1 pancreatic tumor xenografts in athymic nude mice, without side effects. Tumors from capsaicin treated mice demonstrated increased apoptosis, which was related to the activation of JNK and increased cytosolic protein expression of Bax, cytochrome c, AIF and cleaved caspase-3, as compared with controls. Taken together, these results show that capsaicin is an effective inhibitor of in vitro and in vivo growth of pancreatic cancer cells. These findings provide the rationale for further clinical investigation of capsaicin against pancreatic cancer. Ruifen Zhang and Ian Humphreys contributed equally to this work.     

Resistance to the tyrosine kinase inhibitor axitinib is associated with increased glucose metabolism in pancreatic adenocarcinoma

Alterations in energy (glucose) metabolism are key events in the development and progression of cancer. In pancreatic adenocarcinoma (PDAC) cells, we investigated changes in glucose metabolism induced by resistance to the receptor tyrosine kinase inhibitor (RTKI) axitinib. Here, we show that human cell lines and mouse PDAC cell lines obtained from the spontaneous pancreatic cancer mouse model (Kras^G12DPdx1-cre) were sensitive to axitinib. The anti-proliferative effect was due to a G2/M block resulting in loss of 70–75% cell viability in the most sensitive PDAC cell line. However, a surviving sub-population showed a 2- to 3-fold increase in [C-14]deoxyglucose ([C-14]DG) uptake. This was sustained in axitinib-resistant cell lines, which were derived from parental PDAC. In addition to the axitinib-induced increase in [C-14]DG uptake, we observed a translocation of glucose transporter-1 (Glut-1) transporters from cytosolic pools to the cell surface membrane and a 2-fold increase in glycolysis rates measured by the extracellular acidification rate (ECAR). We demonstrated an axitinib-induced increase in phosphorylated Protein Kinase B (pAkt) and by blocking pAkt with a phosphatidylinositol-3 kinase (PI3K) inhibitor we reversed the Glut-1 translocation and restored sensitivity to axitinib treatment. Combination treatment with both axitinib and Akt inhibitor in parental pancreatic cell line resulted in a decrease in cell viability beyond that conferred by single therapy alone. Our study shows that PDAC resistance to axitinib results in increased glucose metabolism mediated by activated Akt. Combining axitinib and an Akt inhibitor may improve treatment in PDAC.